Biomedical subjects

Viji Balasubramanian

Publications and source records attributed to Viji Balasubramanian.

9 recordsLinked to original sources

PubMed2006

Slower onset of low shear stress leads to less neointimal thickening in experimental vein grafts.

Vein grafts respond to low flow and shear stress (tau(w)) by generating thicker walls and smaller lumens through the processes of neointimal hyperplasia and remodeling. Clinically, however, vein grafts with obviously low tau(w), such as those distal to high-grade proximal obstructions, are not infrequently found to be widely patent and pliable. One possible explanation for this phenomenon may be that vein grafts remodel more favorably in response to changes in shear that occur gradually over time compared to abruptly. This hypothesis was tested in an experimental animal model in this report. Two separate models of experimental vein graft failure were created, causing either immediate exposure to ultralow tau(w) (<1 dyne/cm2) or delayed exposure to ultralow tau(w). Under general anesthesia and using a sterile technique, the right external jugular (EJ) veins of 28 New Zealand white rabbits were surgically exposed and isolated. An end-to-side distal EJ/common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. For the immediate exposure group (n = 5), the EJ was suture-ligated just proximal to the thoracic inlet, distal to a small 10-50 microm venous tributary. This created a reversed vein segment immediately and abruptly exposed to high wall tension (2.0 +/- 0.3 x 10(4) dyne/cm) and ultralow tau(w) (0.15 +/- 0.08 dyne/cm2). For the delayed exposure group (n = 22), the EJ was ligated over a 0.035 guidewire, leaving a small aperture to sustain some measure of blood flow and tau(w). This predictably resulted in slightly less wall tension (1.4 +/- 0.2 x 10(4) dyne/cm) and higher tau(w) (0.68 +/- 0.21 dyne/cm2) than the immediate exposure group. During the first week, the small outflow aperture in the delayed exposure grafts thrombosed, eventually exposing them to the same low level of tau(w) as the immediate exposure grafts. Thus, the only difference in the two models was that delayed exposure grafts enjoyed a slower decline in tau(w) than immediate exposure grafts. Fourteen rabbits in the delayed exposure group were harvested over the first 7 days to define the patency curve of the restricted outflow channel. As expected, the small aperture had thrombosed in all animals by 7 days. The remaining 14 grafts were harvested after 4 weeks, and 13/14 remained patent. Examination of the hemodynamic parameters at the time of death confirmed that wall tension and tau(w) had equalized (wall tension 0.9 +/- 0.1 vs. 1.1 +/- 0.1 x 10(4) dyne/cm, tau(w) 0.45 +/- 0.12 vs. 0.30 +/- 0.08 dyne/cm2). Histological examination revealed less neointimal hyperplasia in the delayed exposure group compared to the immediate exposure group (wall thickness 266 +/- 16 vs. 180 +/- 24 microm, p = 0.025) as well as a slightly greater luminal diameter (0.30 +/- 0.02 vs. 0.40 +/- 0.02 cm, p = 0.038). The results of this experiment suggest that slow exposure to reduced tau(w) results in more favorable remodeling (less thickening) than abrupt exposure. This finding may explain the occasional clinical observation of a widely patent vein graft even in the face of proximal arterial obstruction and very low flow; the change in tau(w) presumably occurred slowly mitigating the remodeling response.

Anastomosis, Surgical↗

PubMed2005

Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function.

The vascular endothelium maintains blood fluidity by inhibiting blood coagulation, inhibiting platelet aggregation, and promoting fibrinolysis. Endothelial cells lose these nonthrombogenic properties on exposure to proinflammatory stimuli. We recently identified the Kruppel-like factor KLF2 as a novel regulator of endothelial proinflammatory activation. Here it is found that KLF2 differentially regulates key factors involved in maintaining an antithrombotic endothelial surface. Overexpression of KLF2 strongly induced thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS) expression and reduced plasminogen activator inhibitor-1 (PAI-1) expression. Furthermore, overexpression of KLF2 inhibited the cytokine-mediated induction of tissue factor (TF). In contrast, siRNA mediated knockdown of KLF2 reduced antithrombotic gene expression while inducing the expression of pro-coagulant factors. The functional importance of KLF2 was verified by in vitro clotting assays. By comparison to control infected cells, KLF2 overexpression increased blood clotting time as well as flow rates under basal and inflammatory conditions. In contrast, siRNA-mediated knockdown of KLF2 reduced blood clotting time and flow rates. These observations identify KLF2 as a novel transcriptional regulator of endothelial thrombotic function. The full text of this article is available online at http://circres.ahajournals.org.

Animals↗

PubMed2005

Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.

OBJECTIVE: Vascular remodeling in response to injury or low shear stress (or both) is characterized by neointimal hyperplasia and luminal contraction. When profound, the response leads to restenosis after percutaneous endovascular intervention as well as to de novo stenosis in vein grafts. It has recently been reported that exposure of vein patches to neurovirulence-attenuated Herpes simplex virus-1 (HSV-1) decreases neointimal hyperplasia and increases luminal area. This experiment tested the hypothesis that R7020, a more highly attenuated mutant of HSV-1, would modulate the vascular remodeling response of experimental vein grafts chronically exposed to low shear stress. METHODS: The external jugular veins of 31 New Zealand white rabbits were clamped and intraluminally exposed to vehicle (phospate-buffered saline solution, n = 11), R7020 2.5 x 10(8) plaque forming units [PFU]/mL (n = 8), or R7020 2.5 x 10(9) PFU/mL (n = 12) for 10 or 30 minutes at an average pressure of 80 mm Hg. After exposure, an end-to-side distal external jugular-to-common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. The external jugular was suture-ligated just proximal to the thoracic inlet, distal to a small 10- to 50-microm venous tributary, creating a reversed vein "graft" segment immediately and abruptly exposed to arterial pressure (48 +/- 3 mm Hg) and low shear stress (0.12 +/- .02 dyne/cm(2)). In the 29 animals (N = 31) that survived to harvest, 26 grafts were found to be patent and were analyzed further. Nine grafts were harvested within the first week after operation, snap frozen in liquid nitrogen, and assayed for the presence of the Herpes viral immediate-response protein ICP0 by Western blot analysis. The 17 remaining grafts were perfusion-fixed, excised, stained, and analyzed morphometrically by digital planimetry. RESULTS: In patent grafts, the hemodynamic environment of low shear stress was maintained (shear stress at harvest, 0.26 +/- .06 dyne/cm(2)). Western blot analysis revealed the presence of ICP0 in R7020-exposed vein grafts after 2, 3, 7, and 14 days; ICP0 was not detected in unexposed vein grafts or adjacent carotid arteries. After 4 weeks, vein grafts exposed to R7020 exhibited a statistically significantly increased ratio of luminal radius to wall thickness, indicating altered remodeling (vehicle, 6.7 +/- 1.3; R7020 2.5 x 10(8), 9.1 +/- 1.3; R7020 2.5 x 10(9) ratio, 11.3 +/- 1.4; P < .05 for high dose compared with vehicle). CONCLUSION: A brief exposure of the neurovirulence-attenuated HSV-1 strain R7020 results in an increased ratio of luminal radius to wall thickness in experimental vein grafts chronically exposed to low shear stress.

Animals↗

PubMed2003

Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein.

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.

Alternative Splicing↗

PubMed2003

Sustained inhibition of experimental neointimal hyperplasia with a genetically modified herpes simplex virus.

OBJECTIVE: Reported herein is a potential strategy for sustained smooth muscle cell (SMC) inhibition with a virulence-attenuated herpes simplex virus (HSV). Experiments were conducted in vitro to demonstrate selective SMC cytotoxicity and in vivo to demonstrate reduced neointimal hyperplasia (NIH) in a clinically relevant animal model. METHODS: In vitro: Cultured human umbilical artery smooth muscle cells (UASMC) and venous endothelial cells (HUVEC) were exposed to varying multiplicities of infection (MOI) of a gamma(1)34.5-deleted HSV-1 virus (R849). Cell survival was assessed at 48 and 72 hours with a colorimetric MTT viability assay. In vivo: New Zealand White rabbit external jugular veins (n = 21) were exposed to R849 (2.5 x 10(6) pfu/mL) or culture medium at 110 to 120 mm Hg for 10 minutes, then fashioned as vein patches on carotid arteries. Carotid arteries were ligated distally to decrease blood flow and stimulate a hyperplastic response (ultra-low shear stress model). After 2, 4, 12, and 24 weeks, patched segments were perfusion-fixed with glutaraldehyde and morphometrically examined for NIH formation. RESULTS: In vitro: At 48 hours, R849 exhibited preferential cytotoxicity to UASMC compared with HUVEC, with 11% +/- 10% of UASMCs and 49% +/- 8% of HUVECs surviving after infection with MOI = 25 (P <.05). Higher MOI resulted in poor survival of both cell lines. In vivo: Blood flow was similarly reduced in all animals both at surgery (0.9 +/- 0.1 mL/min vs 1.6 +/- 0.3 mL/min) and at harvest (2.7 +/- 0.4 mL/min vs 2.5 +/- 0.5 mL/min). R849-infected patches exhibited markedly less NIH than control patches did at 2 weeks (162 +/- 14 microm vs 49 +/- 6 microm; P <.05), 4 weeks (190 +/- 27 microm vs 67 +/- 8 microm; P <.05), and 12 weeks (233 +/- 18 microm vs 113 +/- 2 microm; P <.05). CONCLUSION: The virulence-attenuated HSV strain R849 demonstrates selective cytotoxicity for SMC and is capable of sustained inhibition of NIH in an experimental model of vein graft failure.

Animals↗

PubMed2003

Theoretical hydraulic consequences of vein graft taper.

OBJECTIVE: Internal diameter is a strong predictor of patency of infrainguinal vein grafts. However, most vein grafts are tapered, with variable diameter along their length. It is unknown which diameter is most important in determining graft resistive properties, that is, its mean diameter, minimum diameter, or some geometric combination thereof. The purpose of this analysis was to examine the hydraulic consequences of vein graft tapering, with longitudinal impedance (Z(L)), a conduit-specific measure of pulsatile resistance along straight rigid tubes. METHODS: Proximal and distal graft pressure, pressure gradient (DeltaP), and blood flow (Q) were measured intraoperatively in a 100 cm bypass graft and digitally recorded for 10 seconds at 200 Hz. With the Womersley solution for fully developed fluid flow in a rigid tube, a series of DeltaP waveforms were generated for graft diameters ranging from 1.2 to 8.2 mm. With an axisymmetric form of the Navier-Stokes equations, a second series of DeltaP waveforms were computed for grafts with long smooth symmetric tapers ranging from 0% to 90%, with geometric mean diameter of 3.2, 4.2, and 5.2 mm (%Taper = 100 x [proximal diameter - distal diameter]/proximal diameter). For each set of DeltaP and Q, Z(L) was calculated as DeltaP/Q, plotted over a range of 8 Hz, and integrated over 4 Hz to yield integral Z(L). RESULTS: The architecture of the calculated DeltaP and Z(L) waveforms closely approximated their measured counterparts, validating the method. As expected, Z(L) was highly diameter-dependent in a nonlinear fashion. With a clinically relevant boundary of less than 50 x 10(3) dyne/cm(5) as "acceptable," the minimum acceptable diameter of nontapered 100 cm bypass conduits was 4.3 mm. Analysis of graft taper revealed that small amounts of taper in large conduits were well-tolerated. For example, introduction of 32% taper in a 5.2 mm graft (6.2 mm --> 4.2 mm) caused only an 8% increase in integral Z(L) (from 32 to 35 x 10(3) dyne/cm(5)). More pronounced taper in smaller conduits rendered them unacceptable. For example, 53% taper of a 4.2 mm graft (5.7 mm --> 2.7 mm) created a conduit with integral Z(L) of 70 x 10(3) dyne/cm(5), well above the acceptable limit. The relationship between Z(L) and percent taper was nonlinear and strongly dependent on mean diameter. CONCLUSIONS: The relationship between Z(L) and diameter in vein grafts is nonlinear; thus Z(L) increases rapidly in conduits smaller than 4 mm. Tapered vein grafts behave hydraulically like nontapered grafts, provided their geometric mean is greater than 4 mm and their degree of taper is less than 40%. Tapered veins are satisfactory conduits for long-segment bypass grafts, provided their mean diameter is acceptable.

Hemodynamics↗

PubMed2002

Platelets, circulating tissue factor, and fibrin colocalize in ex vivo thrombi: real-time fluorescence images of thrombus formation and propagation under defined flow conditions.

Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1(Alexa.568) (marker for tissue factor), and anti-T(2)G(Cy-5)(1) (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s(-1). A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury.

Antibodies↗

PubMed2002

Local shear conditions and platelet aggregates regulate the incorporation and activity of circulating tissue factor in ex-vivo thrombi.

The presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2,000 s(-1) for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove 'loose' (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl(2) to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (approximately 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s(-1) shear. At 650 s(-1) shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to approximately 1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s(-1) wall shear rate.

Collagen↗

PubMedDate not supplied

Gene therapy for the extension of vein graft patency: a review.

The mainstay of treatment for long-segment small-vessel chronic occlusive disease not amenable to endovascular intervention remains surgical bypass grafting using autologous vein. The procedure is largely successful and the immediate operative results almost always favorable. However, the lifespan of a given vein graft is highly variable, and less than 50% will remain primarily patent after 5 years. The slow process of graft malfunction is a result of the vein's chronic maladaptive response to the systemic arterial environment, its primary component being the uncontrolled proliferation of vascular smooth muscle cells (SMCs). It has recently been suggested that this response might be attenuated through pre-implantation genetic modification of the vein, so-called gene therapy for the extension of vein graft patency. Gene therapy seems particularly well suited for the prevention or postponement of vein graft failure since: (1) the stimulation of SMC proliferation appears to largely be an early and transient process, matching the kinetics of current gene transfer technology; (2) most veins are relatively normal and free of disease at the time of bypass allowing for effective gene transfer using a variety of systems; and (3) the target tissue is directly accessible during operation because manipulation and irrigation of the vein is part of the normal workflow of the surgical procedure. This review briefly summarizes the current knowledge of the incidence and basic mechanisms of vein graft failure, the vector systems and molecular targets that have been proposed as possible pre-treatments, the results of experimental genetic modification of vein grafts, and the few available clinical studies of gene therapy for vascular proliferative disorders.

Arterial Occlusive Diseases↗