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Good Fences Make Good Neighbors: Human Immunodeficiency Virus and Vascular Disease

Abstract Cardiovascular disease, venous thrombosis, and microvascular disease in people with HIV (PWH) is predicted to increase in an aging HIV-infected population. Endothelial damage and dysfunction is a risk factor for cardiovascular events in PWH and is characterized by impaired vascular relaxation and decreased nitric oxide availability. Vascular disease has been attributed to direct viral effects, opportunistic infections, chronic inflammation, effects of antiretroviral therapy, and underlying comorbid conditions, like hypertension and use of tobacco. Although biomarkers have been examined to predict and prognosticate thrombotic and cardiovascular disease in this population, more comprehensive validation of risk factors is necessary to ensure patients are managed appropriately. This review examines the pathogenesis of vascular disease in PWH and summarizes the biomarkers used to predict vascular disease in this population.


INTRODUCTION
Patients with human immunodeficiency viral (HIV) infection have an improved prognosis on antiretroviral therapy (ART) [1]. This is associated with a concomitant drop in the prevalence of opportunistic infections and a corresponding increase in life expectancy [1][2][3]. Noncommunicable diseases have become a major cause of morbidity and mortality in these patients, including large vessel disease (occlusive vasculitides and aneurysms), cardiovascular disease (CVD), and venous thromboembolism [4][5][6][7][8][9][10][11][12][13]. Thrombotic disease is pathological clotting in the vascular system. In arteries, abnormal clotting can result in peripheral vascular disease, myocardial infarction, and cerebrovascular accidents [8]. Venous clots can dislodge and travel to the pulmonary vasculature, a phenomenon known as pulmonary thromboembolism [11]. In the microvasculature system, small disseminated clots can be seen with microangiopathic thrombotic processes, including thrombotic thrombocytopaenic purpura (TTP), TTP-like syndrome, and disseminated intravascular coagulopathy (DIC) [14]. Abnormal clotting in the entire vascular tree occurs in people with HIV (PWH). Thrombotic risk in PWH has been attributed to a number of factors, including the presence of opportunistic infections, prolonged immobility, antiretroviral drugs and other treatments, comorbid conditions (including hypertension and diabetes), and the impact of HIV, itself, on the endothelium [15][16][17]. This review will look at the interaction between HIV, the vascular wall, and pathogenic thrombosis.

THE ENDOTHELIUM
The endothelium is a monolayer of cells that lines the blood vessels [18,19]. It is a highly specialized organ that is responsible for control of both inflammation and coagulation. The endothelium-lining arteries, veins, and capillaries show differential response to stressors that are physiological adaptations to the anatomical location [19]. These stressors include differential shear stress (high in the arterial system and lower in the venous system) that can result in location-specific gene transcription [19].
Under normal conditions, the endothelium is a selectively permeable, anticoagulant surface. It produces a number of molecules that act to limit clotting by inhibiting both platelet activation and coagulation factors (Table 1).
In response to pro-inflammatory stimuli or trauma, the endothelium upregulates cellular adhesion molecules and procoagulant factors and becomes more permeable (Table 2) [19]. This allows leukocytes to translocate across the endothelial surface and into the tissue. There is a local shift from an anticoagulant to a procoagulant surface [27]. Tissue factor may be exposed by trauma, secreted into the peri-endothelial space in endothelial vesicles, or upregulated on leukocytes (especially monocytes) [28,29] and platelets, resulting in activation of the coagulation cascade and clot initiation. Platelets are activated by exposure to subendothelial tissue (primarily collagen) and provide a secondary surface for coagulation, resulting in clot propagation [30].
Following endothelial injury, a number of processes are initiated to repair the damaged endothelium and allow for clot resorption [40]. Bone-marrow-derived endothelial progenitor cells home to sites of vascular injury and stimulate angiogenesis [40][41][42]. Platelets and endothelial cells secrete a number of growth factors (including vascular endothelial growth factor), cytokines (including TGF-beta), and chemokines (including CXCL-12 or stromal derived factor-1) that restore barrier function and stimulate endothelial cell proliferation [43][44][45]. Clot resolution occurs with activation of proteases, like plasmin (through the function of tissue plasminogen activator or tPA present on endothelial cells) [46], which break down fibrin clots with resulting production of fibrin degradation products (measured as D-dimers). This process reduces local hypoxia with stabilization of hypoxia-inducible factor-1, which also can contribute to repair [19].

ENDOTHELIAL DYSFUNCTION
Endothelial dysfunction is a state of aberrant endothelial cell activation that is associated with thrombosis [47]. Endothelial dysfunction classically is associated with arterial atherosclerosis, but it can be more broadly defined to include a prothrombotic state throughout the vasculature [48]. Multiple pathophysiological mechanisms contribute to a phenotype that is characterized by reduced dilatation, decreased arterial compliance, and local inflammation with reduced vascular repair and angiogenesis [19]. Bioavailability of nitric oxide, a key vasodilator and platelet inhibitor, is significantly reduced [25]. The pro-inflammatory mediators that are produced by the damaged endothelial tissue also act directly to upregulate tissue factor expression by leukocytes and to activate platelets [28,49,50]. Cyclo-oxygenase enzymatic activity is upregulated in response to various inflammatory stimuli and results in increased production of prostaglandin E2 and thromboxane A2, which stimulate platelet activation and aggregation [38,51].

Chronic Inflammation Caused by HIV Infection
HIV infection is a cause of chronic inflammation [49]. A detailed description of the causes of inflammation in PWH is outside the scope of this article. Briefly, rapid depletion of CD4 + T cells from the gut-associated lymphoid tissue and, especially, Th17 subsets disrupts the gastrointestinal barrier function with translocation of microbial products across the interface [49,52]. This directly stimulates innate pattern recognition molecules, like toll-like receptors (TLRs), with activation of pro-inflammatory signaling pathways like NFκB. TLRs 7 and 9 also are activated directly by HIV, because low-grade viral replication persists even on therapy [49]. Innate immune effector cells activated through pattern recognition receptors produce pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β [53]. The presence of HIV DNA in the cytoplasm of target cells also activates caspase-1, resulting in increased apoptosis [54]. Chronic inflammation has been associated with functional and quantitative abnormalities in multiple leukocytes subsets. This includes monocyte and neutrophil activation, CD4+ T cell dysfunction and apoptosis, CD8+ T cell activation, and B cell activation [49].
Chronic inflammation results in a state of abnormal endothelial cell activation that has been compared to changes seen in aging [55]. This is characterized by reduced number and function of endothelial progenitor cells with reduced capacity to repair endothelial damage [40,47,56]. Damage to endothelium can result from the release of oxygen-free radicals by activated immune cells, including activated monocytes and lymphocytes. Pro-inflammatory cytokines like TNF-α bind Prostacyclin Platelet inhibition [19] Cluster of differentiation (CD) [39] Scavenges adenosine diphosphate released by activated platelets to inhibit platelet aggregation [20] Endothelial protein C receptor Binds activated protein C to potentiate its activity; complex also acts to protect endothelial cells through the activity on protease-activated receptor-1 and -2 [21] Thrombomodulin Forms a complex with thrombin to activate protein C, which binds and inactivates factors V and VIII (with protein S as a cofactor) [22] Tissue factor pathway inhibitor Inhibits the tissue factor-VIIa complex and factor Xa [23] Primary vasodilator Nitric oxide (endothelium-derived) Mediates vasodilation; inhibits platelet activation [24,25] Endothelium derived hyperpolarizing factor Mediates smooth muscle relaxation and causes vasodilation [26] specific endothelial receptors triggering endothelial apoptosis and activation [50]. Neutrophil activation may result in the production of cytotoxic neutrophils extravasation traps (NETs) that interact directly with coagulation activators and may promote leukocyte adherence [57]. In animal models, neutrophil activation and NETosis is associated with increased risk of thromboembolism [58].

Direct Effects of HIV on Endothelial Cells
The ability of HIV to infect endothelial cells directly is controversial. Small preliminary studies (in vitro) suggested that some endothelial cells could harbor infectious viruses, although larger scale studies have disputed this [59,60]. It is clear, however, that HIV viral proteins can have a detrimental effect on the endothelium with subsequent dysfunction. Tat activates endothelial signaling pathways with downstream reduction in transcription of nitric oxide synthetase and upregulation of monocyte chemoattractant protein-1 (MCP-1) and cell adhesion molecules [61,62]. This promotes both leukocyte activation and leukocyte adhesion to the endothelium. Nef activates pro-inflammatory pathways, including NF-κB and NFAT-1, in both endothelial cells and macrophages, promoting alterations in the monocyte phenotype towards pro-inflammatory cells and release of free radicals that can directly damage the endothelium [63]. In addition, Nef can affect cholesterol transport that predisposes to the formation of foam cells [63]. The HIV envelope proteins, gp-120/41, activate the p38 map kinase pathway that has been linked to increased endothelial permeability, endothelial cell apoptosis, and vasoconstriction [64].

Large Vessel Arteritis and Thrombosis in HIV
Aneurysmal disease and occlusive large vessel disease has been well-described in PWH. Histologically, this disease is pleomorphic with some studies reporting an appearance similar to panarteritis nodosa and others reporting transmural inflammation [65]. The pathogenesis of large cell arteritis is delineated incompletely. Upregulation of chemokine secretion can result in transmural cellular infiltrates with compromise of the vasa vasorum. A suggestion that HIV proteins may mimic arterial proteins with a subsequent cross-reactivity. Aside from direct effects on endothelial cells, studies suggest that arterial smooth muscle cells and fibroblasts may be susceptible to direct HIV infection [65][66][67][68][69]. This may result in weakening of the vascular wall and subsequent dilation [67,70]. This area does, however, require further elucidation.

HIV-Associated Communicable Diseases
HIV-associated immunodeficiency increases the predisposition to and persistence of a number of infections. Chronic infections are associated with a number of changes, including upregulation of procoagulant factors and platelet activation, and mediate a direct effect on endothelial cells. Mycobacterium tuberculosis has been identified in endothelial cells in extrapulmonary infection [71]. Mycobacterial infection is an independent risk factor for thromboembolism and microvascular abnormalities [71,72]. Parasitic infections like Toxoplasma gondii upregulate endothelial adhesion molecules to assist with invasion [73]. In addition, dysbiosis and microbiome perturbations in the gastrointestinal, respiratory, and urogenital tracts occurring in PWH are an independent risk factor for cardiovascular disease [74]. Endothelial cells are targets for Herpesviridae, including cytomegalovirus, Epstein-Barr virus, Kaposi-sarcoma herpes virus (KSHV), and varicella-zoster virus. Persistent infection with these viruses have been associated, especially in aging or otherwise immunodeficient patients, with an increased risk of vasculitis [75] and endothelial dysfunction [76], mediated through inflammatory signaling pathways [73] and through endothelial cell apoptosis [78]. KSHV, specifically, secretes cytokine homologs like viral IL-6 that have been implicated in both mediating a pro-inflammatory milieu and in atypical angiogenesis [79]. Herpes simplex viruses may infect the endothelium with subsequent apoptosis [76]. Finally, other concomitant viral infections (including persistent hepatitis C viral infection) have been linked to increased risk of cardiovascular disease [81,82].

Traditional Risk Factors for Cardiovascular Disease in the ART HIV Era
Traditional risk factors for endothelial dysfunction include the presence of diabetes mellitus, hypertension, and hyperlipidaemia. These conditions contribute significantly to CVD risk in PWH [83,84]. Chronic inflammation (with circulating proinflammatory cytokines) can predispose to insulin resistance through the phosphorylation of insulin receptor substrate-1 [85,86]. Antiretroviral therapy is associated with lipid-abnormalities and dysregulation of glucose-processing pathways, which are independently associated with an increased risk of type 2 diabetes mellitus [85]. Hypertension in PWH is common and a number of potential pathogenic mechanisms have been identified, including lipodystrophy, a pro-inflammatory state associated with the secretion of cytokines and adipokines, and renal disease [87]. HIV protease is molecularly homologous to renin, and renin levels are often inappropriately high [88,89]. Hypertension is exacerbated by worsening endothelial dysfunction [87]. Dyslipidaemia is a common complication of both treated and untreated HIV infection [90,91]. In ART-naïve patients, this may be mediated by direct effects of the virus and the inflammatory milieu. Lipid processing and transportation is altered in PWH. Modified lipids may directly activate pattern recognition receptors [90]. Finally, substance use, especially tobacco use, is increased in PWH [88]. Risk of arterial disease is significantly higher in PWH who smoke and there is a concomitant increased mortality [6,93].

Antiretroviral Drugs
Antiretroviral therapy has reduced the morbidity and mortality of HIV infection, affording improved life expectancy, but long-term therapy can result in endothelial toxicity and vascular dysfunction. This has been linked with a number of metabolic abnormalities, including lipid abnormalities and predominantly an increase in circulating low-density lipoprotein and cholesterol levels [94]. The classes of drugs most commonly implicated are protease inhibitors. The majority of inflammatory and cardiovascular disease biomarkers show a decline with effective therapy, however, confirming a benefit of ART even for cardiovascular disease outcomes [90,[95][96][97].
Few biomarkers have been studied in the ART era in the context of venous thrombosis or microvascular disease, and none have been conclusively linked to diagnosis or prognostication of occlusive vasculitis or aneurysmal disease [65]. This may represent a future study focus.

CLINICAL SCORING SYSTEMS
A number of clinical scoring systems exist to assess arterial, venous, and microvascular thrombosis risk (Table 4). Although thrombosis throughout the vascular tree is described in PWH, relatively few of these scoring systems have undergone validation in this patient cohort. No published performance evaluations of venous thromboembolic scoring systems or microvasculature scoring systems exist in PWH, although small case series have Activates the extrinsic coagulation pathway resulting in thrombin generation [31] Factor VIII Endothelial cells [32] Stabilizes factor IX [32] Platelet activation Collagen and subendothelial matrix Subendothelial tissue [33] Promotes platelet adhesion, activation, and aggregation [33] Cellular adhesion molecules, including p-selectin and e-selectin, ICAM-1, and CXC12 [34] Endothelial cells [33] Promotes platelet adhesion, activation, and aggregation [33] Von Willebrand Factor [35] Weibel-Palade bodies [35] Enables platelet adherence to exposed collagen through its interaction with platelet receptor Ib-V-IX (protects factor VIII from degradation [36,37]) Eicosanoids, including prostaglandins and thromboxane A2 [38] Endothelial cells [38] Promotes platelet aggregation [38] Vasoconstrictive agents Endothelins (predominantly endothelin-1) [39] Endothelial cells, vascular smooth muscles cells, and reproductive system [39] Activates endothelin receptors, increases production of reactive oxygen species, and reduces bioavailability of nitric oxide [39] [128] although systems often either overpredicted [129] or underpredicted [125] cardiovascular risk [123].SCORE generally performed least well [124]. SCORE and D:A:D consistently underestimated cardiovascular risk [117,128] 10-year risk of coronary artery disease only Abbreviations: ADMA, asymmetric dimethylargininine; APC, activated protein C; ART, antiretroviral therapy; AT, antithrombin; â2MI, â2-microglobulin; CD, cluster of differentiation; CIMT, coronary artery intimal medial thickness; CK-MB, creatine kinase; hsCRP, highly sensitive C-reactive protein; LDH, lactate dehydrogenase; LFA-1, leukocyte functional adhesion molecule-1; IL, interleukin; IP10, interferon-ã induced protein 10; LPB, lipopolysaccharide binding protein; Lp-PLA2, lipoprotein-associated phospholipase A2; MCP, monocyte-chemoattractant protein-1; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; PT, prothrombin; sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; TAT, thrombin-antithrombin complexes; TF, tissue factor; TNFR, tumour-necrosis Factor á receptor; tPA, tissue plasminogen activator; VLA-4, very late antigen-4; vWF, von Willebrand factor.  [14,[121][122]. Arterial scoring systems have been more extensively studied. The Framingham risk score, based on the ongoing Framingham heart study, was initially designed to look at heart disease in nondiabetic, Caucasian participants between the ages of 30 and 69 [123]. A number of modifications have been introduced, including an ART regimen specifically for PWH-the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D score) [123]. Other scoring systems that have been evaluated include the systematic coronary risk evaluation (SCORE), atherosclerotic cardiovascular disease risk score (ASCVD), and prospective cardiovascular munster (PROCAM) scores. In PWH, these scoring systems have shown variable performance across validation studies. The Framingham risk score has generally shown the best predictive value for CVD in PWH with D:A:D, with the ASCVD and SCORE systems showing more consistent under-prediction in large European and American cohorts [117,[124][125]. Only smaller cross-sectional evaluations have been undertaken in low and middle-income countries [123,[126][127]. Despite the relatively poor predictive power and data fit shown in many analyses, these scoring systems continue to form the basis of clinical trials in this cohort of patients.
Modeling studies suggest a significant health economic burden of cardiovascular disease in PWH that is predicted to increase as the HIV-infected population ages [136][137]. Understanding the underlying pathogenesis, assessing risk, and identification and validation of appropriate biomarkers will be important. This includes the development of risk scores for microvascular and venous thrombosis. Cardiovascular disease risk is increased in patients who are untreated or who fail to achieve or maintain viral suppression, and early initiation of ART is the mainstay of therapy. In addition, traditional cardiovascular risk factors, including tobacco use, dyslipidaemia, hypertension, and diabetes should be aggressively managed in this population along with chronic infections that can cause chronic inflammation and predispose to vascular disease [138].