Erythrocyte aggregation in sudden flow arrest is linked to hyperthermia, hypoxemia, and band 3 availability.

BACKGROUND: Erythrocyte aggregation is a phenomenon that is commonly found in several pathological disease states: stroke, myocardial infarction, thermal burn injury, and COVID-19. Erythrocyte aggregation is characterized by rouleaux, closely packed stacks of cells, forming three-dimensional structures. Healthy blood flow monodisperses the red blood cells (RBCs) throughout the vasculature; however, in select pathological conditions, involving hyperthermia and hypoxemia, rouleaux formation remains and results in occlusion of microvessels with decreased perfusion. OBJECTIVES: Our objective is to address the kinetics of rouleaux formation with sudden cessation of flow in variable temperature and oxygen conditions. METHODS: RBCs used in this in vitro system were obtained from healthy human donors. Using a vertical stop-flow system aligned with a microscope, images were acquired and analyzed for increased variation in grayscale to indicate increased aggregation. The onset of aggregation after sudden cessation of flow was determined at proscribed temperatures (37-49°C) and oxygen (0%, 10%), and in the presence and absence of 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Both autologous and homologous plasma were tested. RESULTS: RBCs in autologous plasma aggregate faster and with a higher magnitude with both hyperthermia and hypoxemia. Preventing deoxyhemoglobin from binding to band 3 with DIDS (dissociates the cytoskeleton from the membrane) fully blocks aggregation. Further, RBC aggregation magnitude is greater in autologous plasma. CONCLUSIONS: We show that the C-terminal domain of band 3 plays a pivotal role in RBC aggregation. Further, aggregation is enhanced by hyperthermia and hypoxemia.

An in situ inferior vena cava ligation-stenosis model to study thrombin generation rates with flow.

BACKGROUND: Blood flow-induced shear stress affects platelet participation in coagulation and thrombin generation. We aimed to develop an in vivo model to characterize thrombin generation rates under flow. METHODS: An in situ inferior vena cava (IVC) ligation-stenosis model was established using C57BL/6 mice. Wild type C57BL/6 mice were fed normal chow diet for two weeks before experiments. On the day of experiments, mice were anesthetized, followed by an incision through the abdominal skin to expose the IVC, which was then ligated (followed by reperfusion through a stenosis for up to 2 h). IVC blood flow rate was monitored using a Transonic ultrasound flow meter. In sham animals, the IVC was exposed following the same procedure, but no ligation was applied. Thrombin generation following IVC ligation was estimated by measuring mouse plasma prothrombin fragment 1-2 concentration. Mouse plasma factor Va concentration was measured using phospholipids and a modified prothrombinase assay. Blood vessel histomorphology, vascular wall ICAM-1, von Willebrand Factor, tissue factor, and PECAM-1 expression were measured using immunofluorescence microscopy. RESULTS: IVC blood flow rate increased immediately following ligation and stenosis formation. Sizable clots formed in mouse IVC following ligation and stenosis formation. Both plasma factor Va and prothrombin fragment 1-2 concentration reduced significantly following IVC ligation/stenosis, while no changes were observed with ICAM-1, von Willebrand Factor, tissue factor and PECAM-1 expression. CONCLUSION: Clot formation was successful. However, the prothrombin-thrombin conversion rate constant in vivo cannot be determined as local thrombin and FVa concentration (at the injury site) cannot be accurately measured. Modification to the animal model is needed to further the investigation.

Inflammatory monocyte response due to altered wall shear stress in an isolated femoral artery model.

Arteriogenesis (collateral formation) is accompanied by a pro-inflammatory state that may be related to the wall shear stress (WSS) within the neo-collateral vessels. Examining the pro-inflammatory component in situ or in vivo is complex. In an ex vivo mouse femoral artery perfusion model, we examined the effect of wall shear stress on pro-arteriogenic inflammatory markers and monocyte adhesion. In a femoral artery model with defined pulsatile flow, WSS was controlled (at physiological stress, 1.4×, and 2× physiological stress) during a 24 h perfusion before gene expression levels and monocyte adhesion were assessed. Significant upregulation of expression was found for the cytokine TNFα, adhesion molecule ICAM-1, growth factor TGFß, and the transcription factor Egr-1 at varying levels of increased WSS compared to physiological control. Further, trends toward upregulation were found for FGF-2, the cytokine MCP-1 and adhesion molecules VCAM-1 and P-selectin with increased WSS. Finally, monocytes adhesion increased in response to increased WSS. We have developed a murine femoral artery model for studying changes in WSS ex vivo and show that the artery responds by upregulating inflammatory cytokines, adhesion molecules and growth factors consistent with previous in vivo findings.

Nitrosative stress uncovers potent ß2-adrenergic receptor-linked vasodilation further enhanced by blockade of clathrin endosome formation.

This study investigated the effect of sodium nitroprusside (SNP) preexposure on vasodilation via the ß-adrenergic receptor (BAR) system. SNP was used as a nitrosative/oxidative proinflammatory insult. Small arterioles were visualized by intravital microscopy in the hamster cheek pouch tissue (isoflurane, n = 45). Control dilation to isoproterenol (EC50: 10-7 mol/l) became biphasic as a function of concentration after 2 min of exposure to SNP (10-4 M), with increased potency at picomolar dilation uncovered and decreased efficacy at the micromolar dilation. Control dilation to curcumin was likewise altered after SNP, but only the increased potency at a low dose was uncovered, whereas micromolar dilation was eliminated. The picomolar dilations were blocked by the potent BAR-2 inverse agonist carazolol (10-9 mol/l). Dynamin inhibition with dynasore mimicked this effect, suggesting that SNP preexposure prevented BAR agonist internalization. Using HeLa cells transfected with BAR-2 tagged with monomeric red fluorescent protein, exposure to 10-8-10-6 mol/l curcumin resulted in internalization and colocalization of BAR-2 and curcumin (FRET) that was prevented by oxidative stress (10-3 mol/l CoCl2), supporting that stress prevented internalization of the BAR agonist with the micromolar agonist. This study presents novel data supporting that distinct pools of BARs are differentially available after inflammatory insult. NEW & NOTEWORTHY Preexposure to an oxidative/nitrosative proinflammatory insult provides a "protective preconditioning" against future oxidative damage. We examined immediate vasoactive and molecular consequences of a brief preexposure via ß-adrenergic receptor signaling in small arterioles. Blocked receptor internalization with elevated reactive oxygen levels coincides with a significant and unexpected vasodilation to ß-adrenergic agonists at picomolar doses.

Vasoactive effect of fibronectin-derived epiviosamine-1 and related peptides in quiescent and stress models.

OBJECTIVE: Following thermal burn injury, plasma fibronectin degrades within the interstitium; one possible product is EVA-1, PSHISKYILRWRPK found within the FNIII1 . EVA-1 ameliorates thermal burn injury progression, and binds to and enhances PDGF-BB in promoting cell metabolism, growth and survival; shorter related peptides lose these abilities. Here we study the effect of EVA-1 and shorter peptides for their vasoactivity under quiescent and stress conditions. METHODS: Using the hamster cheek pouch intravital microscopy model, five EVA-1 related peptides were applied to small arterioles via micropipette (10-16 -10-4 mol L-1 ) in quiescent tissue and after defined stress: nitric oxide or heat. RESULTS: Peak dilation occurred with nanomolar doses (longer peptides) or below (shorter peptides), blocked by propranolol (beta-adrenergic receptor antagonist). Micromolar doses of the same peptides induced only constriction, not antagonized by phentolamine (alpha-adrenergic receptor antagonist). Scrambled variants of two peptides yielded only constriction, suggesting constriction might be due peptide charge. Each stressor caused a left shift in dilation response, blocked by carazolol. CONCLUSIONS: Thus, this important region of FNIII1 contains sequences that have a gradation of biological functions dependent on the length of the peptide sequence, with increased efficacy for dilation following stressors.

Hyperspectral imaging of nanoparticles in biological samples: Simultaneous visualization and elemental identification.

While engineered nanomaterials (ENMs) are increasingly incorporated into industrial processes and consumer products, the potential biological effects and health outcomes of exposure remain unknown. Novel advanced direct visualization techniques that require less time, cost, and resource investment than electron microscopy (EM) are needed for identifying and locating ENMs in biological samples. Hyperspectral imaging (HSI) combines spectrophotometry and imaging, using advanced optics and algorithms to capture a spectrum from 400 to 1000 nm at each pixel in an enhanced dark-field microscopic (EDFM) image. HSI-EDFM can be used to confirm the identity of the materials of interest in a sample and generate an image "mapping" their presence and location in a sample. Hyperspectral mapping is particularly important for biological samples, where ENM morphology is visually indistinct from surrounding tissue structures. While use of HSI (without mapping) is increasing, no studies to date have compared results from hyperspectral mapping with conventional methods. Thus, the objective of this study was to utilize EDFM-HSI to locate, identify, and map metal oxide ENMs in ex vivo histological porcine skin tissues, a toxicological model of cutaneous exposure, and compare findings with those of Raman spectroscopy (RS), energy-dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Results demonstrate that EDFM-HSI mapping is capable of locating and identifying ENMs in tissue, as confirmed by conventional methods. This study serves as initial confirmation of EDFM-HSI mapping as a novel and higher throughput technique for ENM identification in biological samples, and serves as the basis for further protocol development utilizing EDFM-HSI for semiquantitation of ENMs.

Vasoactive effects of stable aqueous suspensions of single walled carbon nanotubes in hamsters and mice.

Single-walled carbon nanotubes synthesized with iron (Fe-SWCNT) or gadolinium (Gd-SWCNT) show promise as high performance multimodal contrast and drug-delivery agents. Our purpose was to evaluate potential vasoactive effects of SWCNT. Stable aqueous solutions of Fe-SWCNTs or Gd-SWCNTs were made using the biocompatible amphiphilic polymer N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoylsn-glycero-3- phosphoethanolamine (PEG-DSPE). Both aggregated and non-aggregated (sonicated) formulations were tested. The initial vasoactivity of the formulations and their potential for inducing pro-inflammatory endothelial dysfunction were investigated in the hamster cheek pouch and murine cremaster muscle intravital microscopy models. These models provide an assay to test several formulations/dosages in a paired fashion. Abluminal exposure to small arterioles exposes both endothelial and vascular smooth muscle cells. Using abluminal exposures of dosages that would approximate the first pass of an i.v. bolus injection, both Fe-SWCNTs and Gd-SWCNTs were immediately vasoactive. Aggregated formulations induced dilation and non-aggregated formulations induced constriction in both hamsters and mice. Endothelial dysfunction was evident after exposure to either aggregated or non-aggregated forms. General loss of dilator capability was seen after exposure to non-aggregated but not aggregated forms. Thus concentrations mimicking bolus dosing of PEG-DSPE coated SWCNT induce both acute and chronic vascular responses.

In vitro hematological and in vivo vasoactivity assessment of dextran functionalized graphene.

The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ≥ 7 mg/ml, showed a 12-20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1-50 mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect.

Separating Fluid Shear Stress from Acceleration during Vibrations in Vitro: Identification of Mechanical Signals Modulating the Cellular Response.

The identification of the physical mechanism(s) by which cells can sense vibrations requires the determination of the cellular mechanical environment. Here, we quantified vibration-induced fluid shear stresses in vitro and tested whether this system allows for the separation of two mechanical parameters previously proposed to drive the cellular response to vibration - fluid shear and peak accelerations. When peak accelerations of the oscillatory horizontal motions were set at 1g and 60Hz, peak fluid shear stresses acting on the cell layer reached 0.5Pa. A 3.5-fold increase in fluid viscosity increased peak fluid shear stresses 2.6-fold while doubling fluid volume in the well caused a 2-fold decrease in fluid shear. Fluid shear was positively related to peak acceleration magnitude and inversely related to vibration frequency. These data demonstrated that peak shear stress can be effectively separated from peak acceleration by controlling specific levels of vibration frequency, acceleration, and/or fluid viscosity. As an example for exploiting these relations, we tested the relevance of shear stress in promoting COX-2 expression in osteoblast like cells. Across different vibration frequencies and fluid viscosities, neither the level of generated fluid shear nor the frequency of the signal were able to consistently account for differences in the relative increase in COX-2 expression between groups, emphasizing that the eventual identification of the physical mechanism(s) requires a detailed quantification of the cellular mechanical environment.

Curcumin mediates both dilation and constriction of peripheral arterioles via adrenergic receptors.

Curcumin has wound healing attributes mediated through a plethora of biological activities that in general are not ascribed to specific receptors. Recently, we have demonstrated that intravenous administration of curcumin limits burn injury progression in a rat model. As decreased microvascular perfusion is a central element of burn injury progression, we hypothesized that curcumin may induce vasodilation in peripheral arterioles, to improve perfusion. Using mucosal microcirculation as an in situ assay, cheek pouch tissue was exteriorized in anesthetized (phentobarbital 70 mg kg(-1) intraperitoneal) male hamsters (N=60) to observe the terminal feed arterioles (∼8 µm diameter) and the immediately upstream arcade arterioles (∼20 µm). Curcumin (10(-12)-10(-4) mol l(-1)) was applied dose-wise (micropipette, 60 seconds). Subnanomolar curcumin dilated, whereas micromolar doses constricted, the arterioles. For the terminal arteriole: vasodilation logEC(50) -10.3±0.2, peak dilation +39±1%; vasconstriction logEC(50) -8.0±0.4, peak constriction -14±2%. Simultaneous atropine (muscarinic antagonist) or PD142893 (endothelin antagonist) had no effect. Propranolol (ß-adrenergic receptor (ß-Ad) antagonist) enhanced constriction by removing the vasodilation response to curcumin. Phentolamine (α-adrenergic receptor (α-Ad) antagonist) enhanced dilation to curcumin by removing the vasoconstriction response. Thus, the curcumin vasomotor activity on microcirculation was α-Ad and ß-Ad receptor-dependent and its net vasoactive effect was concentration- and time-dependent.

Terminal arteriolar network structure/function and plasma cytokine levels in db/db and ob/ob mouse skeletal muscle.

OBJECTIVE: To investigate the terminal arteriolar network structure and function in relation to circulating plasma cytokine levels in db/db, ob/ob, and their genetic background control, C57/bl6, mice. METHODS: Arteriolar network size and erythrocyte distribution were observed in the resting cremaster muscle (n = 45, pentobarbital 50 mg/kg i.p.). Structural remodeling and inflammatory state were related to 21 plasma cytokine levels. RESULTS: db/db networks were shorter, had fewer branches, and smaller diameters than C57/bl6 controls. ob/ob networks were longer, with similar branch numbers, however with non-uniform diameters. Shunting of erythrocytes to the specific terminal arteriolar branches of the network (functional rarefaction) was prominent in db/db and ob/ob, with further evidence of shunting between networks seen as no flow to 50% of ob/ob arteriolar networks. CONCLUSIONS: Altered levels of plasma cytokines are consistent with structural remodeling seen in db/db, and a pro-inflammatory state for both db/db and ob/ob. Differences in network structure alone predict overall reduced uniform oxygen delivery in db/db or ob/ob. Shunting probably increases heterogeneous oxygen delivery and is strain-dependent.

Downstream exposure to growth factors causes elevated velocity and dilation in arteriolar networks.

Our goal was to characterize changes in flow and diameter with vascular endothelial cell growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF2). Observations were made in arteriolar networks of the cheek pouch tissue in anesthetized hamsters (pentobarbital 70 mg/kg, i.p., n = 45). Local and remote dilation responses to micropipette-applied VEGF or FGF2 yielded similar EC(50) values. The role of gap junctions in the remote response was tested by applying sucrose, halothane or 18αGA to the feed arteriole midway between the remote stimulation and upstream observation sites; all remote dilation to FGF2 was prevented, while only the early dilation to VEGF was blocked. The remote dilation to VEGF displayed a second rheologic mechanism. The second mechanism involved an abrupt increase in upstream velocity and shear rate, followed by nitro-arginine sensitive dilation. To test whether the abrupt increase in shear could be caused by other agents known to cause edema, remote responses to histamine and thrombin were tested. Each caused an abrupt increase in velocity followed by nitro-arginine-sensitive dilation. This study shows that VEGF or agents that increase permeability can initiate an upstream velocity increase with dilation that recruits flow to the network; this is in addition to simultaneous gap junction-mediated dilation.

In vitro biocompatibility of sheath-core cellulose-acetate-based electrospun scaffolds towards endothelial cells and platelets.

Typically, tissue-engineered scaffolds mimic the topographical properties of the native extracellular matrix. However, other physical properties, such as the scaffold mechanical stiffness, are not imitated. The purpose of this study was to fabricate scaffolds with improved mechanical properties and investigate their biocompatibility towards endothelial cells and platelets. To enhance mechanical properties, an electrospinning apparatus was developed that fabricates fibers with sheath-core morphologies. Different combinations of cellulose acetate and chitosan were chosen to modulate the mechanical properties of the formed fibers. We hypothesized that mechanically stiffer scaffolds would improve endothelial cell growth and that all scaffolds would be compatible towards endothelial cells and platelets. Endothelial cell-culture conditions were quantified up to 5 days. Migration onto scaffolds was monitored for 10 days. Platelet aggregation, antagonized by thrombin receptor agonist peptide 6, was measured after scaffold incubation. A platelet activation time-course was assessed with the modified prothrombinase assay. As scaffold mechanical stiffness increased, endothelial cell growth within and adhesion to and migration throughout the scaffolds was promoted. Also, scaffolds did not induce platelet aggregation or activation. These results indicate that the mechanical stiffness of engineered scaffolds regulates endothelial cell-culture parameters and that these sheath-core electrospun scaffolds are compatible towards endothelial cells and platelets.

Uniform-intensity, visible light source for in situ imaging.

A flexible, low-cost, high-brightness light source for biological and biomedical imaging is presented. The illuminating device consists of a custom-size square plastic pouch 10 to 20 mm on a side and 1 to 3 mm thick that can be inserted fully or partially into both in situ or in vitro specimens to be imaged. The pouch contains a silicone-based gel medium embedded with silica particles that scatters light and provides a reasonably uniform, planar light source. Light is delivered to the pouch using a multimode optical fiber and a high-intensity tungsten lamp. Pouch size and geometry can be readily altered as needed for a particular application. Benefits of the device include reasonably uniform light intensity, low temperature rise (<2 degrees C), a nearly white light spectrum, and a thin (<2 mm thick) flexible form factor. The design, fabrication, and preliminary results from the device are presented using hamster cheek pouch tissue, with comparisons to standard intravital microscopy, along with suggestions for further improvement and potential uses.

In vivo validation of biological responses of bFGF released from microspheres formulated by blending poly-lactide-co-glycolide and poly(ethylene glycol)-grafted-chitosan in hamster cheek pouch microcirculatory models.

Microspheres formulated from blending poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-grafted-chitosan (PEG-g-CHN), using a modified in-emulsion-solvent-evaporation method, were investigated for the delivery of protein. A model protein, bovine serum albumin (BSA), was incorporated into the PLGA/PEG-g-CHN microspheres and both initial burst and release kinetics could be modulated by varying the PEG-g-CHN content. Basic fibroblast growth factor (bFGF) was formulated into the microspheres containing 5% PEG-g-CHN and the bFGF contents in the releasates were determined by a receptor-based ELISA with their in vitro bioactivities validated by fibroblast cell culture. The in vivo effect of the bFGF microspheres formulation was evaluated in a hamster cheek pouch model using a 7 day exposure (e.g., before significant vascular remodeling was expected). Using intravital microscopy, the tissue showed no evidence of inflammation with any formulation; deliberate activation of a preconditioning response linked to inflammation was attenuated by BSA microspheres alone. Vasoactive responses (receptor-dependant and independent constriction and dilation) linked to nitric oxide were attenuated, and constriction to endothelin was enhanced in bFGF and not BSA containing microspheres. PLGA/PEG-g-CHN blended microspheres were also demonstrated to be non-inflammatory and non-thrombogenic in vivo by observing the vascular changes in the cheek pouch. In conclusion, the addition of PEG-g-CHN to PLGA microspheres can serve as a sustained delivery vehicle for bFGF and the released protein provides vasoactive changes consistent with chronic bFGF exposure.

Bioassay chamber for angiogenesis with perfused explanted arteries and electrospun scaffolding.

OBJECTIVE: The purpose of this study was to test the hypothesis that explanted perfused arteries can serve as the initial endothelial cell culture source to evaluate the onset of angiogenesis in a cellulose acetate electrospun scaffold. METHODS: Electrospun scaffolds with fiber diameters roughly controlled in three broad ranges: 0.01 to 0.2, 0.2 to 1, and 1 to 5 microm (Nanomed Nanotechnol Biol Med 2:37-41, 2006), were used in cell culture to determine which provides the best culture topology. This scaffold was then tested in a bioassay chamber whose cellular source was an explanted abdominal aorta from donated euthanized mice. Scaffolds were draped over a cannulated vessel perfused for 24 h. Cell viability, density, and morphology were quantified. RESULTS: The largest fiber diameter group provided the best culture topology for human umbilical vein endothelial cells, showing high cell viability and density, and enhanced elongated cell morphology. Addition of single-walled carbon nanotubes decreased cell density significantly but chitosan heightened cell density and promoted spontaneous capillary tube like structure. Viability of endothelial cells increased with higher flow in the bioassay chamber. CONCLUSIONS: Endothelial cells showed a growth preference towards larger diameter fibers. Addition of chitosan improved culture conditions. Thus, this study provides a proof of principle for the possibility of co-culturing tissue engineered vascular networks from a perfused explant.

Remote microvascular preconditioning alters specific vasoactive responses.

OBJECTIVE: The mechanism by which remote microvascular preconditioning (RMP) response is initiated was recently reported (Am J Physiol 290:H264, 2006). The goal of this study was to further characterize RMP and to investigate the extent to which RMP altered local vasoactive responses. METHODS: Arteriolar networks were examined in the cheek pouch of anesthetized (pentobarbital, 70 mg/kg) hamsters (n = 143). RMP was initiated with nitroprusside (SNP) or adenosine (ADO) via micropipette to a downstream arteriole or via tissue bath. Upstream ( approximately 800 microm), at the entrance to the network, local vasoactive responses were obtained with local micropipette application. RESULTS: The RMP response requires 10 to 15 min to manifest, and cycles down with > 5 upstream challenges (local exposures). Without challenge, the response does not cycle down over 8 h. RMP results in enhanced dilation to SNP and attenuated dilation to ADO. SNP can initiate RMP with micropipette exposure to the local downstream arteriolar segment. ADO only initiates RMP with tissue bath exposure, but at low concentrations (10(-8) M). RMP causes a shift in phosphodiesterase (PDE) maintenance of tone, as seen by using PDE inhibitors. This involves a shift from PDE4 to PDE3, and does not appear to affect PDE1 or PDE5. CONCLUSIONS: These findings are consistent with RMP inducing a fundamental shift from cAMP and towards cGMP maintenance of dilatory tone.

Mechanisms initiating integrin-stimulated flow recruitment in arteriolar networks.

Our purpose was to investigate the local mechanisms involved in network-wide flow and diameter changes observed with localized downstream vitronectin receptor ligation; we tested specific K or Cl channels known to be involved in either dilation or elevated permeability following vitronectin receptor activation and tested integrin-linked pathway elements of tyrosine phosphorylation and protein kinase C (PKC). Arteriolar networks were observed in the cheek pouch tissue of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n=86) using intravital microscopy. Terminal arteriolar branches of the networks were stimulated with micropipette LM609 (0.5-10 microg/ml, 60 s) alone or with inhibitors (separate micropipette). Hemodynamic changes (diameter, red blood cell flux, velocity) were observed at the upstream entrance to the network. LM609 alone stimulated first an increase in wall shear stress (WSS), followed by a dilation that recovered WSS to baseline or below. K channel inhibition (glybenclamide, 4-AP) had no effect on the initial peak in WSS, but decreased remote vasodilation. Cl channel inhibition (DIDS, IAA-94, niflumic acid) or inhibition of PKC (chelerythrine) prevented the initial peak in WSS and decreased remote vasodilation. Inhibition of tyrosine phosphorylation (genistein) prevented both. With the use of nitro-arginine at the observation site, the initial peak in WSS was not affected, but remote vasodilation was decreased. We conclude the remote response consists of an initial peak in WSS that relies on both PKC activity and depolarization downstream, leading to an upstream flow mediated dilation and a secondary remote dilation that relies on hyperpolarization downstream at the stimulus site; both components require tyrosine phosphorylation downstream.

Initiation of remote microvascular preconditioning requires K(ATP) channel activity.

The purpose of this study was to investigate vascular preconditioning of individual microvascular networks. Prior work shows that exposure of downstream arterioles to specific agonists preconditions upstream arterioles so that they exhibit an altered local vasoactive response [remote microvascular preconditioning (RMP)]. We hypothesized that mitochondrial ATP-sensitive K+ (K(ATP)) channels were involved in stimulation of RMP. Arteriolar diameter (approximately 15 microm) was observed approximately 1,000 microm upstream of the remote exposure site in the cheek pouch of pentobarbital sodium-anesthetized (70 mg/kg) male hamsters (n = 104); all agonists were applied via micropipette. RMP was initiated by application of pinacidil (Pin), diazoxide (DZ), sodium nitroprusside (SNP), or bradykinin (BK) to the downstream vessel. After 15 min, RMP was apparent at the upstream observation site from testing of local vasoactive responses to L-arginine. Pin, DZ, SNP, and BK each stimulated RMP. To evaluate a specific role for mitochondrial K(ATP) channels in this response, 5-hydroxydecanoate was applied (via a 2nd pipette) during downstream stimulation with agonist. 5-Hydroxydecanoate blocked RMP initiated by Pin, DZ, or SNP, suggesting that mitochondrial K(ATP) channels are involved before SNP signal transduction. To verify this, we applied N(omega)-nitro-L-arginine during DZ or SNP stimulation. RMP was blocked during SNP, but not during DZ, stimulation. Thus stimulation of the RMP response requires mitochondrial K(ATP) channel activity after stimulation by nitric oxide donors.

Ligand-independent activation of vascular endothelial growth factor receptor 2 by fluid shear stress regulates activation of endothelial nitric oxide synthase.

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm2) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.