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In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood. To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.


Introduction
To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus) 1 .
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host 2 . Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood.
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest.
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF 3 . VWF, a shear stressoperational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF 4 .
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.

In Vivo Mesenteric Perfusion Model
1. Preparation/surgery of the mouse 1. Fast the mouse the night before the experiment in order to limit bowel movement. 2. Give a 6-8 week old mouse (C57Bl/6) pre-operative analgesia by a subcutaneous injection of buprenorphine (0.1 mg/kg body weight (BW)) 20-30 min prior to the surgery. 3. Anesthetize the mouse by intra-peritoneal injection of ketamine (125 mg/kg BW) and xylazine (12.5 mg/kg BW). Check by pedal reflex.
Apply vet ointment to prevent dryness. 4. Place the mouse on a thermo-controlled heating pad at 37 °C on a microscope tray. Since this is a terminal procedure, there is no need for strict asceptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle and isolate the jugular vein from the surrounding tissue. 5. Insert a 2 French intravenous catheter into the right jugular vein for infusion of fluorescently labeled bacteria or other solutions. Open the peritoneal cavity via a midline abdominal incision and use cotton swabs to spread the mesenterium and to visualize the mesenteric arteriolar and venular circulation. 6. Place the mouse on the right side on a transparent plate and secure the cannula with tape. Use a hot pack to prevent hypothermia. To prevent dehydration of the tissue, drop 500 µl 0.9 % NaCl on the intestines.
2. Fluorescence microscopy of bacterial adhesion to the mesenteric circulation 1. Work in a dark room. Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope. 2. Topically apply 5 µl of a 10 mM solution of the Ca 2+ -ionophore A23187 dissolved in DMSO. After 10 sec, inject 100 µl labeled bacteria (see step 1) through the jugular catheter. Take time-lapse images. After the experiment is finished, euthanize the mouse according to institutional approved guidelines.
3. Image Analysis 1. Obtain live images using an inverted fluorescence microscope, captured using a black and white camera and developed using any imaging software. Apply automated exposure time and contrast optimization specific to the equipment used. 2. Acquire time-lapse images using the 'Acquisition' tool in the toolbar (Multidimensional Acquisitions -Time) using 40 cycles of 1,000 images/sec. Save the images in an appropriate image file format. 3. Process images using ImageJ analysis software to measure the area of fluorescent signal per image. Define the threshold to set lower and upper threshold values, segmenting gray scale images into features of interest. Identify the region of interest (blood vessel) and measure the area limited to the threshold and the region of interest. Compare bacterial adhesion, expressed as fluorescent area using any statistical or graphing software.

S. aureus adhesion to VWF, subendothelial matrix and endothelial cells is a shear stress dependent phenomenon
To emphasize the role of shear stress in the interaction between S. aureus and VWF, we performed perfusions over VWF coated coverslips at different shear rates (a schematic overview of the in vitro perfusion model is given in Figure 1. Adhesion of S. aureus to VWF increased with increasing shear rates from 250 sec -1 to 2,000 sec -1 (Figure 2), indicating that high shear forces do not inhibit but reinforce the adhesion of bacteria to VWF.
In order to investigate the contribution of VWF to bacterial adhesion to collagen, the main component of the subendothelial matrix, we perfused fluorescently labeled S. aureus over collagen in the presence or absence of VWF. In the absence of VWF, adhesion of S. aureus to collagen The in vitro flow model also allows us to examine the adhesion of bacteria to endothelial cells under flow. We perfused HUVECs with fluorescently labeled S. aureus at shear rates from 500 to 2,000 sec -1 . Where indicated, HUVECs were activated with a Ca 2+ -ionophore, to cause release of VWF. Endothelial cell activation and the subsequent VWF release, increased adhesion of S. aureus (Figure 4A), which formed typical "string-like" patterns of fluorescently labeled bacterial clusters aligned in the direction of the shear force (Figure 4B), suggesting the binding of bacteria along a linear-stretched VWF molecule.
Initial in vivo bacterial adhesion in splanchnic veins is mediated by VWF Since S. aureus is able to adhere to VWF, we used wildtype mice (Vwf

Discussion
Shear stress is a crucial factor for the early bacterial adhesion to the vessel wall and for the subsequent generation of endovascular or endocardial vegetations and metastatic infections 4,5 . We described complementary in vitro and in vivo models to study the pathogenesis of endovascular infections under physiological shear stress. These models have allowed us to identify von Willebrand factor-binding protein (vWbp) as the major S. aureus protein to interact under flow with an injured vascular wall exposing VWF 4 . Endovascular infections, and infective endocarditis in particular, are of concern not only because of sepsis-induced organ failure and death, but also because of local and distant ('metastatic') complications. To cause infective endocarditis and metastatic infections, bacteria have to adhere to the vessel wall and thus resist the shear stress of flowing blood. Most studies on bacteria virulence factors have been performed in static conditions. However, these established interactions might not withstand shear forces and studies under flow conditions can reveal new, previously unrecognized factors in bacteria-host interplay.
Using the micro-parallel flow chamber, we and others have shown the importance of VWF for vascular adhesion. Under shear stress, VWF progressively unfolds from its resting globular structure, and exposes the A1 domain that interacts with platelets via its GPIb receptor 6 . Flow chambers have been extensively used to study platelet function 7 .
Remarkably, also S. aureus adhesion under flow requires VWF, and in particular the A1 domain that is exposed upon shear. We identified vWbp to mediate VWF binding. vWbp is a coagulase that contributes to S. aureus pathophysiology by activating the host's prothrombin. Staphylothrombin, the resulting complex of a bacterial coagulase and prothrombin, converts fibrinogen into insoluble fibrin 8,9 . Our studies have shown that vWbp does not only activate prothrombin, but triggers the formation of bacteria-fibrin-platelet aggregates, which enhance the adhesion to blood vessels under flow 4,10,11 . The in vitro flow chamber model allows to study the different players in bacterial adhesion to cellular or matrix components. Bacterial virulence factors can be studied by using mutants or innocuous bacteria expressing specific surface proteins. Alternatively, pharmacologic inhibitors or blocking antibodies can be added to the medium in the flow chamber. The role of host factors such as different constituents of extracellular matrix can be studied by using coverslips with different coatings. The coverslips can also be covered with endothelial cells, of which the activation status can be modulated by adding specific stimulators. Apart from the vascular wall, the contribution of host blood cells and plasma proteins can Copyright © 2015 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License June 2015 | 100 | e52862 | Page 10 of 10 be studied by adding these factors to the flowing medium. Thus, different conditions of increasing complexity can be studied under standardized conditions of laminar flow to unravel the interactions that allow bacteria to adhere to the vessel wall in vivo.
Interactions identified in the in vitro model are subsequently studied in an animal model to test their relevance in a complex organism. Other in vivo models to study dynamic interactions under flow have been described, such as the hamster dorsal skinfold chamber 12 and the cremaster model 13 . In comparison, the mesenteric perfusion model described here offers several advantages because of its ease of use, the possibility to vary host genetic background of the mice and to evaluate pharmacological interventions.
In conclusion, the described models offer the possibility to study surface proteins not only of S. aureus, but of many other microorganisms in different host backgrounds, to better understand the pathogenesis of vascular infections.

Disclosures
The authors have nothing to disclose.