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Maps of south latin america-Map of South America
South America is the fourth largest continent, the southern of the two continents of the Western Hemisphere. The continent extends c. At its broadest point, near where it is crossed by the equator, the continent extends c. Click on a country for a more detailed map, or try our index. Colombia Ecuador Guyana Paraguay.
It is the tallest mountain found outside of Asia. Highest point in South America is Mount Aconcagua 6, m; 22, ftlocated in the Andes mountain range in Argentina. Argentina Bolivia Brazil Chile. In the extreme south of Argentina, there is a sub arctic climate. If the page is updated in the future, this notice will be removed. Read below to learn before you download the map of Latin America. This is a free download of a vector map of South America that you can use freely for any project you may have:. Other organizations include the Union of South America Nations, which consists of 12 countries and has its headquarters in Quito, Ecuador. Topographic Soutj of South America. Join over 's of others and get Maps of south latin america to my FREE library of.
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Access to your personal information Maps of south latin america restricted. This page was last updated on January off, Seterra on Speedrun. South America: Cities. Oceania: Countries and Territories. By visiting the Site directly or through another site, you Free online illastrated kama sutra the practices described in this Policy. By lstin classifications, South America is seen as a subcontinent of the Americas. Details Not available in any store! It includes Maps of south latin america detailed renderings of nearby countries like Mexicostates like the lower portion of the U. We also may collect information offline, where we also try to protect the privacy of your personal information. I have also Latin American countries that I have personally visited to give you an idea if you are thinking of traveling to Latin America. We also may collect information offline, where we also try to protect the privacy of your personal information. Throughout South America's early history of human inhabitants, it was widely an agrarian society, meaning that its residents survived off of the land, using staples such as fish and beans for food.
Latin America is such a diverse continent.
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- Latin America is such a diverse continent.
South America is found in the western hemisphere. Most of the continent is in the southern hemisphere, although some parts of the northern part of the continent fall in the northern hemisphere. The section in the northern hemisphere includes Venezuela, Guyana, French Guiana, Suriname, parts of Brazil, parts of Ecuador, and nearly all of Colombia.
Sometimes, the dividing line is considered to be the Panama Canal. By some classifications, South America is seen as a subcontinent of the Americas. South America has an area of 17,, km 2 or almost South America is almost twice as big as Europe by land area, and is marginally larger than Russia. South America has coastline that measures around 25, km.
South America has a population of more than ,, with a population density of 21 people per km 2. With Brazil is the largest country in South America, containing 8,, square kilometres. Brazil has a coastline of 7, squared kilometers.
Brazil borders nearly every country in South America, excluding only Ecuador and Chile. The land area of Brazil represents Suriname is the smallest country in South America. The country covers a total of , square kilometres. South America has a total of 12 countries and three dependencies:. A number of trade agreements exist within South America. Mercosur is an international agreement that exists to promote free trade between certain countries on the continent.
Its full members are Argentina, Brazil, Paraguay, and Uruguay. Other organizations include the Union of South America Nations, which consists of 12 countries and has its headquarters in Quito, Ecuador.
The Bolivarian Alliance for the Peoples of Our America is an organization which seeks the social, political, and economic integration of countries in both Latin America and the Caribbean. The topography of South America has been described to resemble a bowl - it has large mountains around its periphery and an interior that is relatively flat.
The continent is mostly made up of lowlands, highlands, and the Andes mountain range, which is the longest mountain range in the world. The Andean Mountains, or Andes, stretch for nearly 7, km across the continent, from the top to the bottom.
The tallest peak in the Andes is Aconcagua in Argentina, which measures 6, It is the tallest mountain found outside of Asia. The Amazon River, which flows from west to east across the north-central portion of South America, is the world's largest river in terms of discharge volume.
By some definitions, the Amazon River is also the longest river in the world - while the Nile River is generally considered to be the world's longest, some studies suggest an alternative source for the Amazon River, which would actually make it the world's longest.
The Amazon Basin Amazonia is covered by the largest tropical rain forest in the world, and running through its heart is the Amazon River and its more than 1, tributaries, seven of them more than 1, miles in length. Measurable rain falls on an average of days a year here, and total rainfall often approaches inches per year. Sparsely populated and positioned high into the Andes of Chile , this somewhat small desert or plateau is a cold place, and one of the few deserts on Earth that doesn't receive any rain.
It's approximately miles wide and miles long. The landscape is totally barren and covered with small borax lakes, lava flow remnants and saline deposits.
The Brazilian Highlights cover about 1,, square miles in eastern, central, and southern Brazil. The highlands have an average elevation of 1, metres above sea level. Due to extensive geographical coverage, the highlands are subdivided into the Atlantic, Southern and Central plateaus each with different climatic conditions as well as flora and fauna. It covers 18, square miles and has a population of over , people. Bananal Island, also in Brazil, is the third largest island in South America, covering 7, South America is home to a range of climate classifications, from equatorial to tundra.
In the north of the continent, near Colombia and Venezuela, weather conditions are generally wet and humid. The same thing goes for areas close to the Equator, including the Amazon Rainforest. Trade winds regulate temperatures in the northeast of the continent, meaning that the climates in Suriname, French Guiana, and Guyana are considerably cooler than their neighbors to the west.
In areas of north Argentina and Paraguay, which fall near the Tropic of Capricorn, rainfall is sparse and temperatures tend to be warm. The west coast of the continent, particularly in Chile, tends to be dry and hot in the summer, with snowfall on the mountains in the winter.
In the extreme south of Argentina, there is a sub arctic climate. As a general rule, the further south you travel, the cooler the temperatures are although temperatures can be quite low in the mountainous regions of the continent as well. South America is extremely biodiverse, with numerous unique species of flora and fauna. Some of the most well-known animals that are unique to South America include the world's largest rodent, the capybara, the world's largest flying bird, the Andean condor, and one of the world's largest butterflies, the blur morpho.
A megadiverse country is a country who is home to the majority of the world's extant species, and has a significant number of endemic species.
There are a number of active territory disputes in South America. Some of these include Guayana Esequiba, which has Guyanese administration but is claimed by Venezuela.
The Gulf of Venezuela is currently claimed by both Colombia and Venezuela. The gulf is an important link between both the Caribbean Sea and Lake Maracaibo, the latter of which contains a large amount of crude oil in its basin. Following a period of urbanization that began in the s, South America today is one of the world's most urbanized continents.
South America is a multilingual continent. By total population, the most popular languages are Portuguese and Spanish. Portuguese is mostly spoken in Brazil, which is the most populated country on the continent. French is spoken in the overseas department of France, French Guiana. Dutch is spoken in Suriname. Guyana is the only country in South America with English as the official language. A significant number of indigenous languages are also spoken in South America.
The most commonly spoken indigenous language is Quechua, followed by Guarani and Aymara. In countries towards the northern part of the continent, such as Peru, Ecuador and even Bolivia, Quechua languages are more common. In Argentina and Bolivia, and especially in Paraguay, Guarani languages are most often spoken.
Other indigenous languages include Aymara, spoken in Bolivia and Peru, and Wayuu, spoken in northern Colombia and northwest Venezuela. Mapudungun is the name of a language spoken in Chile and Argentina. South America is predominantly a continent of Christians.
However, nearly half of the continent's population proclaims itself to be non-practicing. Every country in South America recognizes the separation of church and state. Many of these flags share similarities due to a shared colonial past full of Spanish and Portuguese influence. The flags of Ecuador, Colombia, and Venezuela are particularly similar as they were modelled after the flag of the former state of Gran Colombia.
Some flags, such as that of Bolivia, celebrate indigenous culture. Click on the individual country flag to learn more about its design and meaning. There are some anthropological signs that point back to humans inhabiting South America since at least the year BCE. Throughout South America's early history of human inhabitants, it was widely an agrarian society, meaning that its residents survived off of the land, using staples such as fish and beans for food. Animals like llamas and alpacas were also domesticated around this time.
From this agrarian society, early civilizations were born. Out of the settlements that we are today aware of, the earliest ones were in what is modern day Peru.
In later decades, societies such as that of the Incha civilization and the Mapuche, which actually refers to a diverse group of indigenous residents who lived in what is today known as Argentina and Chile. It was the approval of the Treaty of Tordesillas in that led European explorers to South America. The Treaty of Tordesillas was signed with the intention of allocating land in South America to both Spain and Portugal.
With this treaty, all of the land west of the Cape Verde Islands was to go to Spain. However, due to the lack of precision of the era's geography tools, this distinction was not easily enforceable, nor was it always followed.
This is why the area that we know now today as Brazil was colonized by the Portuguese. Unfortunately, European settlers brought with them diseases like typhus, influenza and smallpox. The native inhabitants of South America did not have the proper immunities to fight these diseases, and many populations perished as a result. As a way to fill the gap in the forced workforce in places like mines, factories, and plantations, enslaved people from Africa were brought over by colonies predominantly controlled by French, Portuguese, and Spanish settlers.
The number of enslaved people brought over from Africa to South America during this time is estimated to be around five million. The 19th century saw a great push for independence and decolonization in South America, with every country having gained independence by The 20th century brought with it military regimes and corruption, as well as a crippling debt crisis. However, it was during this time that South America also underwent a great period of industrialization and advancement.
Today, South America is flourishing. The continent's magnificent natural landscape attracts millions of visitors every year, and tourism numbers continue to increase as international flight connections become more available. Economic expectations appear equally optimistic, as South America is on course to become one of the world's next financial powerhouses.
A current climate of political stability - with a few expectations - also helps secure South America's promising future. Countries Brazil is the largest country in South America, containing 8,, square kilometres. Take our quiz! Geography of South America.
Download printable South America maps. South America is predominantly a continent of Christians. Log in Log out. World: 7 Ancient Wonders. If you submit your email address, we use it to deliver the information to you. One example involves someone calling us to place an order or to ask questions.
Maps of south latin america. Where is South America ?
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Download Now. If you need a blank map of Latin America including Central and South America, feel free to download both maps. This is a free download of a vector map of South America that you can use freely for any project you may have:. If you are looking for the more info on Latin America remember to check out bbc news for the latest on all current events happening in Latin America. Some links in this post contain affiliate links. Meaning, if you click through and make a purchase, book a hostel or sign up for a tour, I may earn a small commission at no additional cost to you.
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Latin America Map - [FREE DOWNLOAD]
South America is the fourth largest continent, the southern of the two continents of the Western Hemisphere. The continent extends c.
At its broadest point, near where it is crossed by the equator, the continent extends c. Click on a country for a more detailed map, or try our index. Colombia Ecuador Guyana Paraguay. Peru Suriname Uruguay Venezuela. Falkland Islands U. Map Index. Argentina Bolivia Brazil Chile. French Guiana Fr.


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DEH 2804 - Clinical Dental Hygiene III
This course is designed to prepare the student to assume a responsible role in the private practice office environment. Emphasis is placed on dental office business systems, and personal and professional development. Private practice dental office visits are included to familiarize the student with various specialty and general dental office procedures and management, and the dental hygienists role in them. Current issues impacting the practice of dental hygiene will also be discussed and debated by the students.
Prerequisites: DEH 1802 and DEH 1802L.
Corequisites: DEH 2804L.
Degrees Offered: A.S.
Credit Hours:1.
Contact Hours: 1.

subset: commoncrawl


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In the news (if you can stand it) ...
I think we might be suffering from news fatigue. It's tempting to give up all communications and just live as though the world isn't going to "you know where" in a hand basket.
Democrats have announced they are launching a formal impeachment inquiry into Donald Trump, calling his conduct regarding Ukraine a "breach of his Constitutional responsibilities."
The inquiry stems from a whistleblower complaint by a member of the U.S. intelligence community recently that Trump may have withheld aid to Ukraine to pressure officials there to investigate the son of political rival Joe Biden.
Trump has agreed to release a transcript of his conversations with the Ukraine president and also the complaint according to news reports.
If all that's not enough, there is the real potential of war with Iran. North Korea has been out of the news lately but you can bet they are not sitting by idly. Same goes for Russia. The trade war with China is another big question mark as is our country's relationship with just about all our allies. Climate change is occurring and half of the population is in denial. The Democrats' hand wringing about impeachment is getting old as is the Republicans' inaction, silence and do-nothing attitude about their leader.
The 2020 Presidential election seems to be on the minds of the politicians mostly and they are doing everything in their power to "hang on to power" or be elected to "power."
To read more please log in or subscribe to the digital edition https://www.etypeservices.com/J.%20V%20Rockwell%20PublishingID250/


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My Wedding Favors – Wedding Tips & Trends – Bridal Blog
PIcnic Wedding
A fall picnic wedding reception on a crisp October day sounds like the perfect wedding theme, right? After a ceremony under the color-changing trees, have your guests meet you in your favorite park for a picnic-style lunch or dinner. They'll wrapped sandwiches, cups of hot cider and plaid blankets while they play yard games. And […]
The post Wedding Theme: A Fall Picnic Wedding appeared first on My Wedding Favors - Wedding Tips & Trends - Bridal Blog.View Post


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Good hunch Aggie-Ocher,
In myth, Doris, "bountiful", was the wife of Nereus and mother of the fifty Nereids.
Dorus (son of Hellen/Ham) can mean either "gift" or "spear". Both of these definitions imply an Osiris typecasting. The Persian Darius was an Osiris also(Azariah/Osorkon). I previously associated "Dorus son of Hellen" with Patriarch Shem, who was thought of as an Osiris of sorts. However, the root Dor/Tor would also relate well to the name Mizraim, "fortifications", derived from the Hebrew matsowr, "tower, strong". The Egyptian name Djoser (equally an Osiris figure) also has the elements d-r-s, although transposed. So, I might have botched the identification of Dorus son of Hellen!
(Note, I also mispelled Dorus as Doris!)
The Roman name Drusus is another matter. It's meaning of "strong" is uncertain and is speculated to derive from the name of a Celtic warrior.
Strength is also connoted in the Hebrew name Azariah/Uzziah (az/uz = strong). Of course strength is also associated with a tower or a bull (taurus).
I wasn't looking for a linguistic path between Drusus/Drusilla and Dorus/Doris. But there seems to be one. It was more than a simple transliteration between languages.
© Charles N. Pope, US Library of Congress. All rights reserved.


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Richard Seay Mccullars
Fayette County High Class of 1971
Personal Info
- Name:
- Richard Mccullars
- Maiden Name:
- Seay
- Graduation Year:
- 1971
Register to view Current location, Gender, Birthday, Relationship Status, Children, Employer, Military, College, and Interests
Memories of Richard Seay Mccullars
What I'm doing now:Register to see what Richard Seay Mccullars is up to now!
Richard Seay Mccullars's Latest Activity:
|- Registered as a FCHS Class of 1971 alumni. July
|- Requested a FCHS yearbook from 1971. July


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The hopes of nations around the world are ignited and extinguished from the blink of any referee's eyes. The feelings and histrionics of one or two athletes can trump the real capabilities of an entire team. The fans themselves get a lot more out-of-manage than the players ever do. Indeed, the supporters are visceral, and once they guess using their guts rather than with their minds, they create availabilities for severe players to gain a plus. Very objective live football recommendations are the type the significant bettors pay attention to.
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As someone who has rehabbed a lot of old houses over the years, I've seen lots of scary electrical "fixes" performed by clueless handymen and homeowners. Some of the perpetrators may even have survived! Even on homes that haven't been butchered too badly, there's frequently a need to add outlets or circuits. Sometimes this is for convenience, so you can run the five-gallon frothing Frappuccino maker simultaneously with the pasta maker and the color-changing mood lighting. Other times it's for more mundane reasons, like meeting code requirements. Whatever the reason, being able to quickly verify that the power is OFF for the circuit you want to work on can be a huge time saver. Using a non-contact voltage tester (NCVT) is an easy way to make sure you really DID power down the right circuit. Klein makes a variety of NCVTs, including some multi-purpose models. They sent one of their latest models our way, so we could see how it measures up. Join us for a full-contact look at the Klein NCVT-6 with built-in laser distance measure. Read More
How To Make A Cool Light Table With The Hot New Ryobi 18V ONE+ Hybrid Soldering Station
This post is sponsored by The Home Depot. Light tables are a staple in many artists' studios. They're great for compositing, iterative drawing, animation, shadow puppets, tattoo artists and showing your friends that gnarly X-ray of the screws in your femur. For a woodworker like myself, it can be a useful tool to create scroll saw and other woodworking patterns. So when we received the model P3100 Ryobi 18V ONE+ hybrid soldering station to try out, I took the opportunity to make one for myself. Trace our steps to find out how you can make a professional quality light table of your own! Read More


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Church Road
Teddington, TW11
£1,250,000 Sold
- 4 bed s
- 2 bath s
- 2 receptions
A beautifully presented four bedroom semi detached Victorian house, which has been skillfully extended to offer almost 1,800 sq.ft of living space over three floors and with the advantage of a garage and parking. The house is situated in a fantastic central Teddington location close to St.Mary's & St. Peters school.
The house is situated in an enviable central location, just 0.4 miles from Teddington station with the fantastic shops and restaurants of the High street equally close to hand. The area is well served by highly regarded local schools with St.Mary's & St.Peters almost on the doorstep.


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Finance Associate
Singular is an international IT company developing one of the most innovative software in the iGaming industry. We continuously strive for improvement by using novel technologies and agile development processes. Founded in 2009, Singular has grown from a start-up of 7 to an enterprise of 100 employees located in 4 offices around the world.
- Providing financial/administrative management oversight of activities including budgeting, forecasting, accounting, invoicing and invoice tracking, cost accounting and cost management, tax planning and tax liabilities and implementation of all services required to financially and administratively support company's operations.
- Help oversee and manage individual accounts
- Ensure payments and follow up on invoices in MK office
- Receive, sort and distribute incoming mail
- Collect and enter data for various financial and accounting spreadsheets
- Reconcile any discrepancies or errors identified by conversing with employees and/or clients
- Adhere to the company's or organization's financial policies and procedures
- Suggest changes or improvements to increase accuracy, efficiency, and cost reductions.
- Operating the cash registry and process the receipts
- Salary calculation and maintaining employee's records
- Creating periodic reports comparing budgeted costs to actual costs.
- Suggesting budgetary changes to increase company profits.
- Working with management at the project level to ensure expense plans are achieved.
- Invoicing clients and following up with all the payments and liabilities in the company.
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- Regular communication with external accountants to secure lawful course of executing things.
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Benefits that we offer:
- Challenging projects
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To apply for this job email your details to email@example.com


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USC hires Mike Bohn, former Cincinnati AD, as athletic director
LOS ANGELES (AP) — Southern California has hired veteran collegiate sports administrator Mike Bohn as its new athletic director.
USC announced the hiring Thursday, nearly two months after Lynn Swann abruptly resigned.
Bohn has spent the past five years as the athletic director at the University of Cincinnati. He also has been the AD at Colorado, San Diego State and Idaho.
The 58-year-old Bohn is the first AD at USC with no ties to the school since 1993. The Trojans' past three athletic directors — Mike Garrett, Pat Haden and Swann — were all former USC football players with no prior experience working in athletic administration, and all three left with the department in turmoil.
Dave Roberts had been USC's interim AD since Swann quit in September.


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In exercise, as well as cancer and ischemia, hypoxia-inducible factor 1 (HIF1) transcriptionally activates hundreds of genes vital for cell homeostasis and angiogenesis. While potentially beneficial in ischemia, upregulation of the HIF1 transcription factor has been linked to inflammation, poor prognosis in many cancers, and decreased susceptibility of tumors to radiotherapy and chemotherapy. Considering HIF1's function, HIF1α protein and its hydroxylation cofactors look increasingly attractive as therapeutic targets. Independently, antioxidants have shown promise in lowering the risk of some cancers and improving neurological and cardiac function following ischemia. The mechanism of how different antioxidants and reactive oxygen species influence HIF1α expression has drawn interest and intense debate. Here we present an experimentally based computational model of HIF1α protein degradation that represents how reactive oxygen species and antioxidants likely affect the HIF1 pathway differentially in cancer and ischemia. We use the model to demonstrate effects on HIF1α expression from combined doses of five potential therapeutically targeted compounds (iron, ascorbate, hydrogen peroxide, 2-oxoglutarate, and succinate) influenced by cellular oxidation-reduction and involved in HIF1α hydroxylation. Results justify the hypothesis that reactive oxygen species work by two opposite ways on the HIF1 system. We also show how tumor cells and cells under ischemic conditions would differentially respond to reactive oxygen species via changes to HIF1α expression over the course of hours to days, dependent on extracellular hydrogen peroxide levels and largely independent of initial intracellular levels, during hypoxia.
The transcription factor hypoxia-inducible factor (HIF) plays a critical role in the mammalian response to oxygen (O2) levels. HIF1, the first characterized member of the HIF family, transcriptionally activates hundreds of genes associated with angiogenesis in cancer, exercise, and ischemia, as well as energy metabolism, nutrient transport, cell cycle, and cell migration (85, 98).
HIF1α and HIF1β make up the HIF1 heterodimer. The β-subunit is constitutively expressed in cells. Expression of the α-subunit may be induced by a number of pathways, and its degradation is highly sensitive to O2 levels. Called a master switch for hypoxic gene expression (76, 85), intracellular HIF1α in normoxia is experimentally undetectable; during hypoxia, it rapidly accumulates in the cell nucleus and triggers gene expression. Molecular players involved in this process have come to light over the past 6 years; research has begun to define roles for prolyl hydroxylases, iron, ascorbate, hydrogen peroxide, 2-oxoglutarate, succinate, and von Hippel-Lindau protein in the HIF1 pathway.
Concomitantly, the study of reactive oxygen species (ROS) and the interest in antioxidants as potential dietary supplements for prevention of cancer, cardiac dysfunction, and neurodegeneration has grown rapidly. Ongoing debate surrounds the role of these compounds in hypoxic responses and the utility in pursuing them as preventative therapeutics. Some studies have shown increased ROS expression in hypoxia (10, 40), while others show a decrease (33, 96). Increased HIF1α expression has been found to contribute to mitochondrial activity (1), and specifically ROS formation, during hypoxia (26, 40, 81). However, other studies have demonstrated a decrease in HIF1α with increasing ROS (22, 99). Finally, some studies have shown no effects of H2O2 (87) or mitochondrial ROS in general (96). Related observations seem nearly as conflicting. Under hypoxic conditions, mitochondrial complex III may produce ROS, and the presence of high ROS concentrations generated from the mitochondria has been shown to stabilize HIF1α (8, 9, 20, 26). On the other hand, ROS may be produced in the cytosol, derived from NADPH oxidases (17, 33), and ROS may play a larger role in HIF1α expression during normoxia than hypoxia (43).
There are several hypotheses as to how ROS interact with the HIF1 pathway and alter HIF1α expression (recent related reviews include references 41 and 75). One possibility is that hydrogen peroxide oxidizes ferrous iron (Fe2+) to its ferric form (Fe3+), prohibiting the necessary binding of ferrous iron to the HIF1α hydroxylation enzymes, prolyl hydroxylases (PHDs) (71). Another change could be in the recruitment of ascorbate as a free radical scavenger, preventing ascorbate from reducing ferric iron and/or preventing ascorbate from binding directly to the PHDs. If ROS increased rather than decreased free Fe2+, as suggested by some experiments, HIF1α hydroxylation would instead increase (56). Additionally, 2-oxoglutarate (2OG) and succinate (SC) are also compounds involved in HIF1α hydroxylation whose concentrations could be altered by free radicals and mitochondrial dysfunction (38, 56, 71). A fourth mechanism by which ROS could influence the HIF1 pathway is through changing the availability of oxygen to bind directly to the PHDs or changing PHD phosphorylation.
To address these alternate mechanisms and analyze possible competing factors involved in pro- and antioxidant therapy in cancer and ischemia, we developed a computational model describing the in vivo system and used it to observe dynamics currently inaccessible at the molecular level in vivo. Experimentally, ROS have been shown to affect the HIF1 pathway through changes in H2O2, Fe2+, Asc, 2OG, or SC levels (61, 71), and mechanisms involving these compounds were the focus of this study.
The model consists of kinetic equations mapping the molecular steps in HIF1α degradation in normoxia, HIF1α synthesis in chronic hypoxia, and effects of the enzyme and cofactors involved in the HIF hydroxylation pathway. Kinetic values were estimated from in vitro studies, and results were validated by comparison to a series of independent experimental data. The input is cellular oxygen level, and the output is HIF1α levels in the nucleus in relation to necessary intermediate reactions, including reactions with prolyl hydroxlylase, iron, 2-oxoglutarate, ascorbate, succinate, and von Hippel-Lindau (VHL) ligase. The model was expanded to represent two possible mechanisms of how ROS interact in the HIF1 pathway: (i) at high concentrations, ROS induce HIF1α by decreasing the activity of prolyl hydroxylases, and ROS effects can be silenced by antioxidants; (ii) in some cells with damaged mitochondria, the opposite effect (ROS decreasing HIF1 activity) is possible through increased iron and 2-oxoglutarate, cofactors in HIF1α degradation.
Using this model, we demonstrate how ascorbate, iron, hydrogen peroxide, 2-oxoglutarate, and succinate would alter HIF1α expression in two representative hypoxic microenvironments: cancer cells and cells during ischemia. We show how these compounds affect adaptation to chronic hypoxia, taking into account possible changes in succinate and reactive oxygen species levels associated with increased anaerobic metabolism and oxidative phosphorylation, such as those found in cancer. Results offer insight into the pro- and antioxidant effects of five compounds present in the HIF1 pathway and how they differ in a tumor microenviroment compared to ischemia. The model demonstrates temporal-specific molecular mechanisms that could be harnessed for use in cancer prevention, recovery from ischemic injury, and repression of angiogenesis and inflammatory signaling regulated by HIF1α expression.
Formulation of the computational model.A model of oxygen sensing by HIF1α was introduced and validated elsewhere (77). Here, we built an HIF1 computational model to incorporate potential mechanisms of ROS and antioxidants reacting within the HIF1α pathway. We represented ROS through two distinct means: (i) changes in the cofactors involved in HIF1α hydroxylation and (ii) the addition of hydrogen peroxide. The first mechanism is through direct increases or decreases in the concentration (availability) of certain cofactors of the hydroxylation pathway, i.e., by altering [Fe2+], [Asc], and [2OG] levels, independent of H2O2 concentrations. The second mechanism focuses on H2O2 as a representative ROS and affects HIF1α hydroxylation through Fe2+ and a Fenton reaction.
The complete model, based on an extensive analysis of experimental data, includes the hydroxylation of HIF1α by PHDs and the ubiquitination of hydroxylated HIF1α by VHL. Table 1 lists the compounds relevant to the current model. Equation 1 describes the overall scheme of HIF1α degradation. Equations 2 and 3 depict the oxidation of iron by reaction with hydrogen peroxide and the reduction of iron by ascorbate, respectively. The complete model includes HIF1α hydroxylation, independent reactions of iron and ascorbate, hydrogen peroxide production, succinate accumulation and product inhibition, PHD2 synthesis and HIF1α synthesis in chronic hypoxia, and the binding of HIF1α to VHL.
The scheme of the overall biochemical reaction of HIF1α hydroxylation, with succinate product inhibition is as follows: (1) Iron oxidation by reaction with hydrogen peroxide is depicted as follows: $$mathtex$$\[\mathrm{Fe}^{2{+}}{+}\mathrm{H}_{2}\mathrm{O}_{2}{\rightarrow}\mathrm{Fe}^{3{+}}{+}\mathrm{OH}^{{\cdot}}{+}\mathrm{OH}^{{-}}\]$$mathtex$$(2) Iron reduction by ascorbate (2, 101) is depicted as follows: $$mathtex$$\[4\mathrm{Fe}^{3{+}}{+}2\mathrm{Asc}{+}\mathrm{O}_{2}{\rightarrow}2\mathrm{dehydroAsc}{+}4\mathrm{Fe}^{2{+}}{+}2\mathrm{H}_{2}\mathrm{O}\]$$mathtex$$(3) Enzyme-substrate binding kinetics are used to describe the hydroxylation reactions. Governing equations are determined from mass balances for the substrate and the intermediate enzyme-substrate complexes. A combination of enzyme-substrate saturation assumptions was used for the binding of iron, ascorbate, 2-oxoglutarate, and oxygen to PHD2, PHD2 hydroxylation of HIF1α, and VHL-mediated ubiquitination. In the hydroxylation reaction of PHDs with HIF1α, we represented the binding of PHD2 with the substrates iron, 2-oxoglutarate, and oxygen sequentially; the redox reactions for ascorbate and iron are included as separate equations (77). These hydroxylation steps and their output were validated against experiments previously (77, 78). Model inputs are initial compound concentrations, including cellular O2 levels (Table 2). The output is HIF1α levels in the cell cytoplasm. The described kinetic model includes all previously studied reactions (77, 78).
Three feedback loops, HIF1 autocrine upregulation, HIF1 induction of PHD2, and succinate product inhibition, were determined to govern HIF1 levels and regulate the response to chronic hypoxia (Fig. 1) (78). Production terms were included for the synthesis of HIF1α and PHD2 at 3 and 4 h of hypoxia, respectively. The PHD2/HIF1α synthesis ratio is 0.01:0.05 in all cases presented in this paper; this ratio was chosen by estimating that between 4 and 8 hours there is a sixfold increase in HIF1α compared to initial conditions. C1, a constant in the synthesis terms for PHD2 and HIF1α, was set at 0.1 (78).
The effects of succinate were represented by one of two mechanisms, as previously described (78). Briefly, product inhibition by succinate was included by modifying the backward kinetic rates for the PHD2 complex binding to unhydroxylated HIF1α. This inhibition could result from 2-oxoglutarate being converted into a succinic acid salt or succinate and therefore not allowing 2OG to be available to the PHD2 forward hydroxylation reaction. As an alternate possibility, succinate accumulation could trigger a change in PHD2 activity or HIF1α levels independent of product inhibition, through a yet-unknown signaling mechanism. A hypothesis is that succinate accumulation and related tricarboxylic acid (TCA) cycle changes correlate to a redistribution of intracellular oxygen or changes in oxygen demand. There is not yet experimental evidence to rule for or against this hypothesis about succinate's role; there is potentially related work on nitric oxide's involvement in intracellular oxygen redistribution (44) and structural evidence linking ROS and succinate dehydrogenase (45, 104) that leave this as a possibility to explore. One way of modeling this potential change is by altering the oxygen available for hydroxylation as a function of succinate production. In the figures shown below, we refer to the two mechanisms as "production inhibition" and "SC signals O2 depletion."
In the present study, the model was expanded to include the production, transport, and metabolism of hydrogen peroxide, a representative ROS that is overproduced by mitochondria malfunction and during ischemic stress (Fig. 1). Here we only present equations not included in our previous work.
Hydrogen peroxide.Hydrogen peroxide is a reactive oxygen species that is also involved in the oxidation of Fe2+, a cofactor in HIF1α hydroxylation (equation 3). Changes in the concentration of hydrogen peroxide were incorporated into the model by a mass balance to represent H2O2 production (either release from the cell mitochondria during hypoxia or NADPH oxidase-dependent H2O2 production), H2O2 degradation by glutathione peroxidase and catalase, and influx from the surrounding extracellular environment. H2O2 degradation and production were represented in all cell types through additional kinetic terms. The kinetic equation for hydrogen peroxide production and degradation is as follows: $$mathtex$$\[\frac{d[\mathrm{H}_{2}\mathrm{O}_{2}]}{d\mathrm{t}}{=}p{-}k_{\mathrm{catalase}}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{free\ catalase}]{-}k_{\mathrm{GPx}}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{GPx}]{-}k_{\mathrm{Fe}3}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{Fe}^{2{+}}]\]$$mathtex$$(4) where p is a constant production term, estimated from experimental data in different microenvironments. This includes the oxidation of Fe2+ by H2O2. H2O2 degradation is dependent on concentrations of catalase and glutathione peroxidase, GPx. Catalase is a mitochondrial enzyme that degrades H2O2 to water in a catalytic reaction (and in a peroxidatic reaction, it degrades H2O2 in the presence of a hydrogen donor; here we consider only the catalytic reaction) (25). Glutathione peroxidase is a second enzyme that controls intracellular H2O2 levels, also by consuming H2O2 to produce water; unlike catalase, it is predominantly active in the cytosol. The kinetic rates kcatalase, kGPx, and kFe3 are apparent first-order rates for a given initial H2O2 concentration. Table 2 provides the initial concentrations for H2O2 under different conditions, as well as estimates for the kinetic term p and pseudo-first-order rate constants for the terms kcatalase × [free catalase] and kGPx× [GPx]. kFe3 was estimated as 1.1 × 10−2 μM−1 min−1 (12, 62). Table S1 in the supplemental material shows relative catalase and GPx activities for a number of cancer cell types, compared to noncancerous tissue. These values lend additional weight to the assumption that there is a threefold difference in catalase activity between tumor and noncancerous conditions (59).
To complete the representation of H2O2 intracellular concentration, possible diffusion of H2O2 into and out of the cell needs to be addressed. The H2O2 mass balance including transport is as follows: $$mathtex$$\[\frac{d[\mathrm{H}_{2}\mathrm{O}_{2}]}{d\mathrm{t}}{=}p{-}k_{\mathrm{catalase}}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{free\ catalase}]{-}k_{\mathrm{GPx}}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{GPx}]{-}k_{\mathrm{Fe}3}[\mathrm{H}_{2}\mathrm{O}_{2}][\mathrm{Fe}^{2{+}}]{+}\frac{p_{\mathrm{membrane}}{\cdot}A}{V}\ {\cdot}\ ([\mathrm{H}_{2}\mathrm{O}_{2}]_{\mathrm{extracellular}}{-}[\mathrm{H}_{2}\mathrm{O}_{2}])\]$$mathtex$$(5) where [H2O2]extracellular is the extracellular concentration of hydrogen peroxide. The last term of equation 5 includes the change in [H2O2] from the extracellular environment into the cell (units of μM·min−1), {(pmembrane·A)/V}·[H2O2]extracellular and {(pmembrane·A)/V}·[H2O2], the outward H2O2 flux from the cell. Equal permeability is assumed for flux into the cell and flux out of the cell. From experimental analysis using T cells suspended in vitro, an estimate of the permeability coefficient for H2O2 across cell membranes, pmembrane, is 2 × 10−4 cm·s−1. If the cells are represented as spheres with area A of ∼627 μm2, radius of 7.5 μm, and volume V, then pmembrane·A/V ≈ 0.8 s−1 or 48 min−1 (5).
In representing nonischemic, noncancerous cells in vivo, where intracellular and extracellular H2O2 are at low levels, transport into and out of the cell is neglected, and the mass balance includes only production and metabolism (25), as in equation 4. For other conditions, including transport, the mass balance is given by equation 5. H2O2 diffuses rapidly, and for the current model, H2O2 concentration is represented as uniform throughout the cell.
In vivo concentrations of H2O2.While in vivo intracellular H2O2 concentrations in most cell types and in humans are not yet precisely known, estimates from measurements in bacteria and rat livers indicate levels on the order of 0.2 μM. In Escherichia coli, intracellular H2O2 concentrations were measured as 0.13 to 0.25 μM by one study (37) and estimated to be lower than 20 nM in the absence of exogenous sources in another study (82). Levels for mammalian cells range from 10−3 to 10−1 μM, depending on H2O2 production rates (25). Maximal H2O2 concentrations used for signaling in mammalian cells have been estimated as 0.5 to 0.7 μM (88). Variability among steady-state intracellular H2O2 in a single cell type is anticipated to be 50% or less (under different conditions of superoxide dismutase expression, relative steady-state intracellular H2O2 varied less than 20% in hamster lung fibroblasts [93]). However, it also should be noted that H2O2 concentrations can rise as high as 100 μM in phagocytes (72), and viable, transient levels of H2O2 in brain cells of >200 μM have been proposed (7).
The effects of added extracellular H2O2 have been studied in vitro by many research groups. As one example, extracellular H2O2 concentrations of 0.01 to 0.25 mM were shown to induce changes in characteristics of the potassium current in endothelial cells (higher concentrations of up to 0.5 mM had different effects on the potassium channel potential) (21). A transport model of H2O2 and experiments on H2O2 consumption indicate that following a change in H2O2, an equilibrium is reached within seconds between intracellular and extracellular H2O2 levels, with a 7- to 10-fold higher concentration in extracellular space, dependent on membrane permeability (5).
For the purposes of this study, initial in vivo intracellular concentrations of H2O2 were estimated from experiments, for normal physiology, as well as cancer and ischemic microenvironments (Table 2). For tumor cells, noncancerous (normal) cells, and noncancerous cells in ischemia, the default initial concentrations for H2O2 were 0.2 μM, 0.02 μM, and 0 μM, respectively. In the model, extracellular H2O2 is estimated as 1.4 μM, approximately sevenfold greater than the initial intracellular level for tumor and ischemic cells, and 14 μM during reperfusion (Table 3) (5, 88). Experimentally determined relative values for H2O2 kinetic parameters are provided in Table 5, below, for specific tumor types.
Numerical solution.The system of nonlinear differential equations was solved using Mathworks Matlab software. The ode23s solver, based on a modified Rosenbrock formula, was used to find a solution for the series of 19 differential equations. For the time integration, the solver used adjustable time steps with a default absolute error tolerance in the solution of 10−6 μM.
Tumor microenvironment.The tumor microenvironment is predominantly associated with hypoxia, high rates of glycolysis, and high levels of reactive oxygen species. Furthermore, select enzymes that degrade ROS (e.g., catalase for H2O2) appear in lower levels in tumor-bearing mammals than in healthy ones (19, 25). Results of the model show effects of ROS on the HIF1α pathway in tumors through two proposed mechanisms: (i) direct changes in concentrations of the hydroxylation cofactors Fe2+, Asc, and 2OG, or (ii) introduction of elevated levels of H2O2 and changes in H2O2 metabolism, production, and transport.
ROS in tumors represented through Fe2+, Asc, and 2OG.Experiments suggest that ROS interact with the HIF1α pathway by altering the availability of Fe2+ and Asc, hydroxylation cofactors (22, 34, 38, 54). The model showed effects of high ROS levels on a hypothetical microenvironment by changes in Fe2+ and Asc levels (Fig. 2A). Where [Fe2+]0 and [Asc]0 were upregulated, the peak amount of HIF1α occurred within the first hour, as in transient hypoxia (see Fig. S1A in the supplemental material). Without ROS and baseline levels of Fe2+ and Asc, the model predicted a maximum in unhydroxylated HIF1α concentration near 4.5 h, whereas when ROS were represented by a decrease in [Fe2+]0 and [Asc]0, the time to maximum HIF1α expression shifted by several hours, and HIF1α remained elevated over several days (Fig. 2A). Tenfold decreases in [Fe2+]0 and [Asc]0 led to an approximately fivefold increase in maximum HIF1α expression (Fig. 2A).
Other experiments indicate that Fe2+ and 2-oxoglutarate are released by damaged mitochondria. 2-Oxoglutarate, like succinate, is a product of the citric acid cycle. In hypoxia, it has been hypothesized that nitric oxide causes mitochondrial dysfunction and thereby 2OG and Fe2+ upregulation mediated by the reactive oxygen species peroxynitrite (56). Increased intracellular 2OG and Fe2+ then contribute to increased hydroxylation of HIF1α by the PHDs. The model showed effects of high ROS levels on a hypothetical microenvironment by changes in Fe2+ and 2OG levels (Fig. 2B).
ROS in tumors with succinate effects.Certain tumors, such as pheochromocytomas and paragangliomas, are associated with mitochondrial mutations and deficiencies in the enzyme succinate dehydrogenase (SDH). Succinate and 2-oxoglutarate are also intermediate products in the citric acid (TCA) cycle. In the hydroxylation reaction, PHD2 simultaneously splits oxygen to hydroxylate HIF1α and oxidizes and decarboxylates 2-oxoglutarate to succinate (83). Downregulation of SDH, an enzyme that degrades succinate to fumarate, leads to intracellular succinate accumulation, HIF1α stabilization, and HIF activation (83). Through in vitro and in vivo experimental observations, succinate was hypothesized to act as a product inhibitor of the PHD hydroxylation reaction of HIF1α (55, 74, 78, 83). Deciphering the relationship between succinate and ROS has been the subject of numerous studies, as well. Because of the structure of SDH redox centers, mutations in SDH have been predicted to result in ROS formation (104). Studies using both Caenorhabditis elegans (48, 86) and tumors with SDH mutations (49) have shown increased ROS. However, other studies have provided evidence that ROS is not necessary for succinate accumulation to produce a pseudo-hypoxic effect and stabilization of HIF1α (64, 74, 84). Shedding light on both possibilities occurring under different conditions, recent research showed a link between mutations in particular subunits of mitochondrial complex II (succinate-ubiquinone oxidoreductase B [SdhB]), ROS production, and HIF1α activation, and no ROS connection with other complex II mutations (SdhA) (42). Another relevant study showed that SDH mutations could specifically contribute to tumor formation via both ROS production and a proliferative response associated with succinate accumulation (91). The computational model's predicted effect of ROS on tumors with SDH deficiency is shown in Fig. 2C; Table 4 shows the initial concentrations used to represent conditions found in vivo (78). ROS, represented by a 10-fold decrease in initial Fe2+ and 2OG concentrations, shifted the time to the peak accumulation of HIF1α by several hours (Fig. 2B). The time to peak HIF1α accumulation was shifted up to ∼48 h, when the model was used to represent in vitro conditions of succinate product inhibition (see Fig. S1B and C in the supplemental material; in these figures, [SC]0 is 500 μM, the maximal concentration used to represent SDH deficiency in published in vitro experiments) (83). ROS, represented by a 10-fold decrease in initial Fe2+ and Asc, also shifted the time to peak accumulation of HIF1α (see Fig. S1D in the supplemental material).
ROS in tumors represented through H2O2.The effect of ROS on HIF1α could be solely related to one ROS species, H2O2, its initial concentration, and its production in tumor cells (61). Measurements of H2O2 in vivo are difficult to obtain, and estimates for a base value of 0.2 μM in tumors were obtained from experiments, as described above. Fibroblasts that became tumorigenic with the expression of Nox1, the catalytic subunit of an NADPH oxidase, expressed H2O2 at a level 10-fold that of normal fibroblasts (6), in agreement with a number of studies that had established the relationship between increased H2O2 and tumorigenicity (24, 30, 90). Results from the model show the effect of an increase in initial [H2O2]0, H2O2 production, and H2O2 metabolism predicted for a tumor cell (Fig. 3 and 4) and? a tumor cell with SDH deficiency represented by two distinct mechanisms (Fig. 4; see also Fig. S2 in the supplemental material). Additionally, the effects of extracellular H2O2 on HIF1α are predicted and compared to available in vitro experimental data (see Fig. S4 in the supplemental material).
Ischemia microenvironment.The ischemic microenvironment is associated with hypoxia, and high levels of reactive oxygen species are found both following the ischemic insult and during reperfusion.
ROS in ischemia represented through H2O2.The mechanism of ROS involvement in HIF1α expression during ischemia could be solely related to H2O2 initial concentrations and production. If this is the case, the model predicts a temporal expression of HIF1α in cells exposed to ischemia, similar to that of tumor cells (Fig. 3). This expression changes at the onset of reperfusion, which induces a larger amount of H2O2 production and alters H2O2 transport (Fig. 5; see also Fig. S4 in the supplemental material). If H2O2 extracellular levels remain elevated, the resulting increase in HIF1α lasts, uninhibited by any mechanism currently modeled, whether the intracellular conditions are approximated as hypoxia or normoxia (see Fig. S4 in the supplemental material).
ROS in ischemia represented through H2O2, Fe2+, and Asc or 2OG.Using the ischemia microenvironment, a third and a fourth mechanism for how ROS affect the HIF1α pathway were tested, with increases in H2O2, Asc, and Fe2+ or increases in H2O2, 2-oxoglutarate, and Fe2+ simultaneously. The model results for changes in Fe2+ and Asc or 2OG are shown, in conjunction with increased production of H2O2 (Fig. 6). In comparison to the Fe2+ and Asc mechanism of ROS effects (a 10-fold increase in Fe2+ and Asc) (Fig. 6A), an increase in 2OG accelerates the production of HIF1α and elevates its expression much higher by 8 h of hypoxia (Fig. 6B).
The variable effects of ROS on HIF1α can be attributed to three main factors: (i) the degree of hypoxia, (ii) the form and intracellular location of ROS produced, and (iii) the molecular microenvironment of the cell. The third was the focus of this model. Before discussing the relevance of the microenvironment, it is worthwhile to briefly describe the roles of the first two factors, as they relate to the presented results.
ROS, O2 levels, and ROS species.ROS production requires oxygen, so it is not surprising that ROS are expressed in different concentrations during anoxia, hypoxia, and normoxia. However, a direct correlation between ROS levels and O2 availability remains elusive. Low O2 limits formation of superoxide and its by-products (33), while hypoxia also has been shown to increase ROS, possibly through release by the mitochondria electron transport chain (41). Equally intriguing, ROS appear to play distinctly different roles in anoxia, hypoxia, and normoxia, with respect to the HIF1 system. In anoxia, the electron transport chain in mitochondria may serve as oxygen sensor and regulator of HIF1α expression (41); as oxygen consumption by mitochondria becomes limited by O2 availability only when O2 falls below ∼0.1%, mitochondria would not likely be an effective detector of moderate hypoxia. In hypoxia, some experiments suggest that at low or intermediate ROS concentrations induced by a superoxide generator, HIF1α is downregulated, as PHD activity is upregulated (22, 54). In related experiments, during normoxia, high ROS levels increased HIF1α, by blocking PHD hydroxylation. Additional studies have also indicated that in long-term hypoxia (12 h), the effect of ROS is to signal HIF1α degradation and downregulate HIF1α expression (27). In contrast, other experiments have shown in hypoxia that ROS upregulated HIF1α (6, 40, 75).
One explanation for the conflicting effects of ROS on HIF1α may be that HIF1α expression is dependent on particular reactive oxygen species, and not others (20); experiments differ in how they have measured ROS and in what cell types. ROS is produced by several means, which will affect ROS location and signaling. In normal cells, mitochondrial complex I and III and cytosolic monoamine and NADPH oxidases produce H2O2. In cancer cells, ROS, and H2O2 in particular, are additionally overproduced by mitochondrial respiration, while in ischemia, cells may be susceptible to ROS from both mitochondrial dysfunction and the extracellular environment, including effects of reperfusion injury. In both cases, inflammatory cells may also release ROS locally and affect intracellular levels in tumors and ischemic cells. Of all reactive oxygen species, H2O2 seems to be the one with the noticeable effect on the HIF1 system (20, 34, 61). However, the reactive oxygen species peroxynitrite may serve as an oxygen donor during HIF1α hydroxylation, as well as mediate Fe2+ and 2OG release from the mitochondria (56, 89), and effects of superoxide on HIF1α have been shown in renal carcinoma (46) and renal medullary interstitial cells (103).
Predictions: ROS and the cellular microenvironment.The molecular environment, the focus of this work, distinguishes why the effects of ROS differ between tumor cells and ischemic cells. In tumor cells, the duration of hypoxia is generally longer, and the cell adapts to anaerobic metabolism and likely relatively stable levels of ROS production. HIF1α levels peak, as in normal cells, before 12 h (Fig. 4), and do not become elevated again until several days (see Fig. S3A in the supplemental material). In contrast, for the ischemic cell, the duration of ischemia is often hours or less, and cell is starved of oxygen but unable to adapt as readily as a cancer cell; additionally, the levels of ROS rapidly increase following the infarct or occlusion, and with perfusion, and then remain elevated for days, eventually decreasing (Fig. 5; see also Fig. S4A and B in the supplemental material).
ROS mechanisms.We represented the effects of ROS on the HIF1α through two different mechanisms: (i) altering the relative concentrations of cofactors in PHD2 hydroxylation of HIF1α and (ii) H2O2 production, transport, and metabolism. Both mechanisms involve the availability of free ferrous iron (Fe2+), a cofactor in the hydroxylation reaction, via a mechanism that has been shown in experiments (34). They differ in that the first mechanism was modeled by changing the availability of Fe2+ and Asc (Fig. 2A and 6A) or Fe2+ and 2OG (Fig. 2B and C and 6B), while H2O2 only altered the availability of free ferrous iron in the model. Changes in H2O2 concentration were highly dependent on the microenvironment (Tables 2 and 3).
The results from the model lend credence to the hypothesis that ROS interacts with the HIF1 system through several different mechanisms, and they may help explain conflicting experiments on HIF1α expression and ROS (Table 5). If ROS work by both increasing Fe2+ and Asc or 2OG (Fig. 2 and 6) and increasing H2O2 (thereby decreasing free Fe2+) (Fig. 3, 4, and 5), depending on the hypoxic conditions, then there could be a dual effect of ROS so that in some cases it upregulates HIF1α and in others it downregulates it. Moreover, in the first mechanism, Fe2+, Asc, and 2OG are quickly depleted in ischemic-reperfusion conditions, and the relatively lower HIF1α level is a transient effect (Fig. 6). Beyond 5 to 12 h (with a 10-fold increase in 2OG and Asc, respectively), HIF1α levels reach the same level or higher, as they do using H2O2 alone to represent ROS (Fig. 6; see also Fig. S4B and C in the supplemental material). Depending on the duration of hypoxia in experiments, the model then predicts the same ROS conditions would yield either a relative upregulation or downregulation of HIF1α. Model results using H2O2 as the representative ROS are in agreement with experimental studies showing H2O2 depletes ascorbate (70) (data not shown). The effect of an infinite source of Asc and free Fe2+ has not yet been measured experimentally or modeled with respect to the HIF1α system and ROS; this would be an interesting test of altering hypoxic response through nutritional supplementation.
ROS in cancer and in ischemia. (i) H2O2 in cancer.Restricting the effect of the ROS in the model to changes in H2O2 levels offers insight into how differences in cancer and ischemic microenvironments determine distinct cell fates. The effects of H2O2 on the HIF1α concentrations are dependent on the dose of H2O2, levels of hypoxia, and cell type. Seemingly contradictory, high levels of H2O2 are associated not only with the progression of cancer to metastasis but also the susceptibility of cancer cells to cell death (61). An existing hypothesis to explain this observation is that all cells, cancer cells included, have an upper threshold for H2O2 concentration, above which apoptosis occurs and below which cells proliferate at a rate proportional to the amount of H2O2 (36, 59) (Fig. 7). The model predicts cancer cells reach a lower steady-state level of H2O2 than cells exposed to ischemia and reperfusion (Fig. 8). Assuming the threshold hypothesis is true, the model implies cancer cells are more apt to survive in their hypoxic microenvironment well beyond the survival time of cells exposed to ischemia-reperfusion, at the same duration of hypoxia.
Additionally, HIF1α expression is increasing as H2O2 increases or remains elevated below the threshold (Fig. 7), making the cancer cells more virile and less susceptible to radiation and chemotherapy (50). Increased HIF1α expression triggers the production of angiogenic factors in both cancer and ischemic cells, but the ischemic cells have less of a chance to survive their high intracellular ROS levels, while angiogenesis fuels the cancer cells' proliferation. If ROS works through an increased Fe2+, Asc, or 2OG mechanism too, the time to peak HIF1α levels is delayed (Fig. 2 and 6; see also Fig. S1 in the supplemental material), potentially delaying the onset of angiogenesis. Furthermore, there may be a threshold for maximum HIF1α levels like that hypothesized for H2O2 levels, above which apoptosis occurs, in which case, again the cells exposed to ischemia-reperfusion would be more susceptible than cancer cells (Fig. 7B). In support of the model's predic-tion of extracellular H2O2 effects on HIF1 in cancer cells, results were compared to experimental data looking at in vitro conditions where cancer cells were supplemented with H2O2 and insulin. While no direct comparison can be made, as the conditions are not the same and the experiment assessed HIF1 activity through hypoxic gene activation, the trend of increasing HIF1 activity and then saturation at high extracellular H2O2 agree (see Fig. S4 in the supplemental material).
(ii) H2O2, ischemia, and reperfusion.Along with differentiating the effects of ROS in cancer cells and cells exposed to ischemia, the model results shed light on two additional experimental observations: concentration-dependent effects of H2O2 and the extent of reperfusion injury. The model described H2O2 concentration as a function of production, transport, and metabolism. The intracellular concentration of H2O2 (high or low) rapidly reaches an equilibrium level dependent on extracellular concentrations. This is a function of its quick transport across the cell membrane (5, 16), combined with the driving force of intracellular metabolism (see Fig. S2B and S3D in the supplemental material). As a consequence of the rapid equilibrium, initial concentrations of H2O2 are of minimal relevancy, and the model predicts that sustained intermediate levels of intracellular H2O2 have the most significant effect on increasing HIF1α expression in tumors and ischemia and altering the hypoxic response long term (Fig. 7A). While there may be many explanations other then sustained, elevated intracellular H2O2 levels, this conclusion also offers an interesting perspective on experimental evidence showing that intermediate H2O2 levels produce more DNA lesions than higher or lower H2O2 concentrations (69) and supports experiments showing some cells can fully recover from transient very high levels of H2O2 (7).
The model predicts reperfusion produces a far greater increase in H2O2-dependent HIF1α expression than ischemia alone (Fig. 5), even while H2O2 production decreases to normal levels with reperfusion after 3.5 h (Table 2). This is true even if reperfusion is considered to provide enough oxygen for cells to reach normoxia (see Fig. S3A and B in the supplemental material). Anticipated high extracellular levels of H2O2 during reperfusion drive this elevated H2O2-dependent HIF1α (Table 3).
The model then highlights how regulating hydrogen peroxide temporally is essential in treatments for ischemia-reperfusion injury. In vivo, high oxidant stress following ischemia may drive angiogenesis, allowing recovery of normoxia through the growth of vessels, while on the other hand too high levels of oxidants damage tissues. Modeling is key to correctly pinpointing the effective time frame for therapeutics, and as the computational modeling advances with new experimental and clinical measurements, antioxidant and proangiogenic treatments could be tailored to individuals.
Metabolism of ROS: catalase, GPx, and variability.In experiments using human hepatoma cells, catalase overexpression did not show an appreciably different effect on HIF1 expression or transcriptional activity (87). The opposite has been shown in other cell types (20). The authors of the first study suggested that H2O2 played little or no role as a signaling molecule in the hypoxic response. As another possibility, which could explain discrepancies between studies, H2O2 may play a role in HIF1 activation only or predominantly in hypoxia, but not anoxia (81). Our model suggests a third explanation. An increase in intracellular catalase concentration does not have a strong effect on the hypoxic response via direct HIF1 activation, while extracellular H2O2 and catalase levels do. Lending support to the model's finding that extracellular H2O2 greatly determines the cellular hypoxic response, recent studies have indicated catalase added to cell medium has a strong effect on cell-cell communication in microglia via NO signaling (47). Furthermore, another study showed that an adenovirus containing catalase affected the activity of apoptosis signal-regulating kinase 1, another signaling molecule involved in hypoxia and ischemia, whose activity is dependent on H2O2 and indirectly related to HIF1 (57).
The model offers plausible mechanisms to explain several phenomena associated with ROS effects on the HIF1 system; however, it is worthwhile to mention limitations of the current model. In vivo intracellular H2O2 concentrations depend on a number of parameters that vary by cell type. These parameters include the concentration and activities of the peroxisome enzyme catalase and glutathione peroxidase, predominant in the cell cytosol; both enzymes degrade H2O2 into water. The concentrations and activities of these enzymes were approximated as constants in the model (Table 2), and further studies would represent how known changes in their activities affect intracellular H2O2 concentrations and the HIF1 system. For example, the concentration of catalase is known to vary with cell type, ranging from 4 ng/106 cells in lymphocytes to 850 ng/106 cells in macrophages (80). The variability among endothelial cells is expected to be less; however, it has not been established experimentally to our knowledge (in bacteria, a variability in catalase activity of 10- to 20-fold has been assessed [79]). Studies measuring tissue-level catalase activity lend weight to the model's assumption of constant catalase activity during ischemia and reperfusion (39). However, the concentration and activity of intracellular catalase, as well as the scavenging enzyme GPx, are likely specific to the cell and tissue type, as well as age (67) (see Fig. S1 in the supplemental material). H2O2 membrane transport has been modeled here as unlimited diffusion (65), while facilitated transport by aquaporins may also occur (15). Additionally, extracellular H2O2 is estimated as a constant for each condition considered in the model. When more observations become available on the temporal changes in extracellular H2O2 during cancer and ischemia, it would be interesting to include effects of changing local extracellular H2O2.
The model predicts that in tumors, the metabolism of H2O2 drives increased transport from the extracellular microenvironment, while H2O2 production by a single cell is a fraction of what is being metabolized (see Fig. S3D in the supplemental material). The model approximated uniform metabolism and production throughout the cell, while production occurs in subcellular units or areas of the cytoplasm. As another consideration, the transport of H2O2 into and out of the cell and intracellular membranes (i.e., peroxisomes) affects the H2O2 gradients present in the cell. How the response to an isolated, high concentration of ROS differs from a cell's response to uniform intracellular ROS elevation merits exploration.
Another characteristic of the model suggests an avenue for experimental pursuit. The current model has no specific feedback on ROS effects. Model predictions for HIF1α accumulation in chronic conditions correlate well with a range of experiments showing a peak in HIF1α before 12 h (Fig. 3; see also Fig. S2A in the supplemental material). However, without a way to negatively regulate ROS, for hypoxic and ischemic durations beyond 48 h, the model predicts elevated HIF1α levels that have not been seen in many experiments (see Fig. S2A and S3B and Table S2 in the supplemental material). Potential sources of adaptation to ROS or downregulation of ROS effects on HIF1α could be changes in PHD2 activity or O2 redistribution.
A systems view of HIF1α signaling.As HIF1 is a transcription factor, its protein regulation is interesting in the greater context of systems biology. From experiments and computational modeling thus far, in chronic hypoxia HIF1α has two positive autoregulatory feedback mechanisms, HIF1α upregulating itself and the hydroxylation product succinate downregulating PHD2 to upregulate HIF1α, and one negative one, HIF1α-dependent upregulation of PHD2 (78). Furthermore, ROS regulates HIF1α expression; this provides another form of autocrine regulation by a hypoxic cell.
What does a hypoxic cell gain from these multiple regulatory pathways, both negatively and positively? Negative autoregulation alone speeds the response time for a closed regulatory circuit and leads to saturation in the protein. If HIF1α only regulated PHD2, and not itself, this would be the expected result; on the other hand, if HIF1α regulated HIF1α alone and not PHD2, the positive feedback would lead to a slower response time and could lead to indefinitely increasing HIF1α conditions. From experiments, HIF1α auto-upregulation occurs an hour or more prior to HIF1α-dependent PHD2 upregulation. One might expect from this a characteristic peak found in HIF1α expression, with a chronic decay rate of HIF1α dependent on the synthesis ratio of PHD2:HIF1α (78). The staggered response coupling both positive and negative feedback then potentially optimizes both the degree of HIF1α upregulation and the speed of its regulation during periods of chronic hypoxia.
HIF1α protein regulation during hypoxic durations of less than 3 hours is modulated without feedback. This provides a response within minutes (HIF1α accumulation in hypoxia and HIF1α oxygen-dependent degradation). There is no need to keep the response on, once oxygen is restored. It may also be that some genes are upregulated by HIF1 specifically during long-term hypoxia, and the concentration or duration of the transient HIF1 response is insufficient to trigger their activation.
To summarize and weigh current hypotheses on ROS at a systems level, a brief network motif analysis of possible ROS mechanisms in regards to HIF1 and hypoxia is presented (Fig. 8). Figure 8 provides circuit representations of hypotheses of ROS and HIF1α interactions. A coherent feed-forward loop refers to a circuit where the indirect path and the direct path yield the same response; an incoherent loop refers to one where the two paths cause opposite effects (3). Of the three proposed mechanisms of ROS, the incoherent feed-forward type 1 (Fig. 8B), is one of the most prevalent circuits in biological systems, albeit at the gene level (3). In this case ROS may increase HIF1α through limiting O2 availability, ferrous iron, and Asc or decrease HIF1α by increasing 2OG and Fe2+ or donating O2 in hypoxia, all cofactors in PHD2 hydroxylation. If ROS mechanisms can be described by this circuit, pulse-like dynamics in HIF1α expression may be possible; ROS would begin to upregulate HIF1α independently of PHD2 and then PHD2 would be upregulated by ROS, leading to HIF1α downregulation (3). Other possible mechanisms of ROS interactions are shown, as well as the effects of succinate and hypoxia globally on HIF1α. At the transcriptional level, these configurations are rarer than the incoherent feed-forward type 1, and they are characterized by limited function.
If the prevalence of specific motifs at the transcriptional level can be extrapolated to molecular species and proteins, the presented circuit analysis helps support the hypothesis from the molecular model that ROS acts by both up- and downregulating HIF1, rather than one or the other. The allure of representing the system in circuit diagram form is both enhanced and tempered by the known biological complexity of the HIF1 system. In graphical form, hypothesized positive and negative controls on HIF1 become simplified; adding components, e.g., metabolic pathways, becomes relatively easy. However, even while highlighting characteristics of simple circuits and network motifs, the interactions, delay time, protein versus gene response, and multiple connections can alter the excepted benefits or characteristics of a particular motif.
The presented model demonstrates several molecular mechanisms for how ROS signaling can affect the HIF1 pathway through Fe2+, Asc, 2OG, SC, and H2O2. We showed how tumor cells and cells exposed to ischemia would differentially respond to ROS via changes to HIF1α expression over the course of hours to days. Model results also show that in hypoxia (both in cancer and ischemic microenvironments), H2O2 intracellular levels rapidly reach equilibrium with extracellular levels, largely independent of initial intracellular levels. H2O2 transport and metabolism, more than cellular production, dictate HIF1α levels. Antioxidants (e.g., Asc) can alter the amount of ROS that is metabolized intracellularly and restore free Fe2+ levels, but unless the extracellular ROS levels change too, this effect is transient. Applied to therapeutic manipulation of hypoxic response, model results imply that antioxidants would need to be applied judiciously at the correct intervals (a sustained, moderate level) to have a noticeable effect on HIF1α levels either in the cancer or ischemic-reperfusion microenvironment. The optimal concentration would be dictated by the hypoxic microenvironment, and the model suggests noticeably higher doses would be needed to avoid reperfusion injury than those needed to prevent cancer cell proliferation or reduce ischemic damage alone.
This work was supported by NIH 1F32HL085016-01 (A.Q.) and NIH HL079653 and NIH HL087351 (A.S.P.).
We thank J. Pouyssegur for useful discussions.
- Received 11 January 2008.
- Returned for modification 20 February 2008.
- Accepted 30 May 2008.
↵▿ Published ahead of print on 16 June 2008.
↵† Supplemental material for this article may be found at http://mcb.asm.org/.
- American Society for Microbiology


text: (Bloomberg) -- Investors can't quit emerging markets, but they're becoming a lot more picky..After the benchmark index for developing-nation stocks entered a bear market this year, some analysts and strategists are recommending buyers avoid the broad-based gauges to focus on specific countries offering the best opportunities. While in the past buying an index and letting it ride produced fat returns, investors now need to be more selective..Ardra Belitz, a Lazard Asset Management money manager who leads the emerging-income team, favors countries with predictable, credible policy regimes.."I continue to find value in Indonesia, as well as Mexico, where policy makers were quick to react to the challenging external backdrop," she said in an email Oct. 3. "Colombia's fiscal and external accounts are supported by higher energy prices, and select frontier countries" are also appealing..READ: Feel-Good Factor Returns to Emerging Markets, With a Caveat (Sept. 27).John Normand, a strategist at JPMorgan Chase & Co., recommends going long duration where rates are high or rising, such as Mexico and Indonesia. He favors currencies from countries with large current-account surpluses like Thailand, oil exporters with reduced political risk like Colombia, and those where rates may rise, such as the Czech Republic and Poland.."The case for regional and country differentiation rather than index investing is building," JPMorgan strategists led by Normand wrote in a note published Sept. 28, citing their projection that the U.S.-China trade conflict would move into an end-game in the first quarter of 2019. JPMorgan doubled down on that call on Oct. 3 when it cut China stocks to neutral from overweight, citing trade-war risks..Argentina and Turkey are often cited as the two riskiest emerging-market countries at the moment. The peso has lost more than half its value this year, and the lira is down 38 percent, amid concern that central bank policies aren't adequately addressing current-account deficits in both countries.."Markets still seem to be more selective now and have clearly singled out Argentina and Turkey as the weakest links," ABN Amro economists Arjen van Dijkhuizen and Nora Neuteboom wrote in a note Oct. 3..All this means investors may want to pay more attention now than they have in the past, and that simply hopping into an exchange-traded fund or a ready-made basket of assets may not work as well as it sometimes has. The MSCI Emerging Markets Index jumped 34 percent last year, for example.."So far in 2018, a handful of countries have had an outsized impact on headlines and on sentiment," Lazard's Belitz said. "Country-specific drivers (sanctions in Russia with the risk of more, a loss of confidence in Argentina, political uncertainty in Brazil, tariffs/political risks/high private sector foreign debt rollover ratios in Turkey, etc.) are likely to continue to influence results.".To contact the reporter on this story: Joanna Ossinger in New York at email@example.com contact the editors responsible for this story: Chris Nagi at firstname.lastname@example.com, Brendan Walsh, Dave Liedtka.©2018 Bloomberg L.P.


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VLFCU is proud to have Schild's IGA as a business member. Originally established in 1946 as Schild's Food Town in Monroeville, this second-generation, family-owned grocery store has been a part of the Norwalk community since 1955. Many locals have fond memories of George Schild, who welcomed everyone into his store and knew just about everyone's name!
Today, owners Dave and Marion Schild, continue that tradition of making everyone feel welcome. Dave started working in the store at the age of 12, earning $.95/hour. He grew up in the business, and after working construction in California for a few years, he knew where he belonged. He returned to Norwalk, fell in love with Marion, and both worked at the store through the 1980s until 1994 when they took over the business.
If you have never been in Schild's IGA, we suggest that you stop in for a pleasant surprise. From the outside, the store looks impressive, but it's when you get inside that you feel the difference. The store is filled with wonderful aromas mingling together from the bakery and deli. The smell of Charley's Chicken was literally mouth-watering! According to Dave, customers come in every day for the made-from-scratch doughnuts and hot homemade lunches that are available to carry out or eat-in at their café.
This friendly family is all about serving their customers and providing "easy solutions," as Marion puts it. Schild's IGA really is a one-stop-shop that offers a floral department, fresh-cut meats, Ohio Lottery, a full-service deli, and a Sunoco Gas Station. You can even earn fuel point rewards to save up to $.50 per gallon, up to 15 gallons per fill-up. The gas station is also one of just a few local providers offering E85 ethanol fuel. The store also offers online ordering for pick up or delivery.
Schild's IGA gives back to the community through local sponsorships and community "give-back" opportunities. This November, customers can help those in need by donating IGA items to be given to a local food pantry. Every May, Schild's hosts an Anniversary Sale during which special sales and giveaways are offered. You can learn more about their store, lunch specials, and services by visiting their website and following them on Facebook.
Located at 171 Milan Avenue in Norwalk, OH, Schild's IGA and Sunoco Gas station are open from 6:00 am to 9:00 pm seven days a week. Whether you're a Norwalk local or just passing through, it's worth stopping by to be greeted by the wonderful and caring employees who make you feel like one of the family.


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Kate's StoryTree is a space made of people, stories and toys. It is a museum and a library and a great big art cupboard. But ultimately it is a weekly story time group for families to immerse themselves in a different story, with props and activities in an entertaining session.