Li-ming Gan

A New Computerized Biomechanical Perfusion Model for ex vivo Study of Fluid Mechanical Forces in Intact Conduit Vessels

We have developed a new computerized biomechanical ex vivo perfusion system for intact conduit vessels in which a wide range of combinations of intraluminal pressure, fluid flow and shear stress could be set and maintained at target levels in mammalian conduit vessels under controlled metabolic conditions. Mean wall shear stress is calculated using the formula:τ = 1/2 * (ΔP/L)3/4 * (8ηQ/Π)1/4.Accuracy of the wall shear stress calculation was validated by ultrasonographic imaging of the vessel radius. In a series of simulation experiments, the hemodynamic homeostasis functions of the system were challenged by generating a wide range of vascular resistance in artificial vessels and by pharmacologically induced changes in vascular tone in intact human vessels. Despite rapid changes in vessel resistance, shear stress and pressure, or flow and pressure were maintained well at target levels. Shear- and pressure-stimulated production of the vasodilator prostaglandin E2 (PGE2) was used to validate the biological relevance of the model. PGE2 release was significantly more stimulated by high (25 dyn/cm2) compared to low (<4 dyn/cm2) shear (ANOVA, p = 0.012). High compared to low intraluminal pressure depressed the production of PGE2 (ANOVA, p = 0.019). In summary, the computerized perfusion model appears to offer new possibilities of investigating the complex interplay between fluid mechanics and the vascular wall.

Elevated Intraluminal Pressure Inhibits Vascular Tissue Plasminogen Activator Secretion and Downregulates Its Gene Expression

We recently discovered that patients with essential hypertension have a markedly impaired capacity for stimulated release of tissue plasminogen activator (tPA) from vascular endothelium. This defect may reduce the chance of timely spontaneous thrombolysis in case of an atherothrombotic event. We now investigated whether increased intraluminal pressure as such may depress vascular tPA release or downregulate its gene expression. Segments of human umbilical veins were studied in a new computerized vascular perfusion model under steady laminar flow conditions for 3 or 6 hours. Paired segments were perfused at high or physiological intraluminal pressure (40 versus 20 mm Hg) under identical shear stress (10 dyne/cm(2)). Quantitative immunohistochemical evaluation of cellular tPA immunoreactivity was performed on paraffin-embedded 5-microm vascular sections. tPA mRNA in endothelial cells was quantified with reverse transcription real-time TaqMan polymerase chain reaction with GAPDH as endogenous control. Secretion of tPA into perfusion medium was evaluated with SDS-PAGE and Western blotting, followed by densitometric quantification. High-pressure perfusion downregulated tPA gene expression with a 38% decrease in tPA mRNA levels (P=0.01) compared with vessels perfused under normal intraluminal pressure. tPA release into the perfusion medium was markedly suppressed by high pressure (P<0.01 ANOVA). The intracellular storage pool of tPA was reduced after 6 but not 3 hours. Thus, elevated intraluminal pressure downregulates tPA gene and protein expression and inhibits its release from the endothelium independently of shear stress. The defective capacity for stimulated tPA release that we demonstrated in patients with essential hypertension might thus be an effect of the elevated intraluminal pressure per se.

Temporal regulation of endothelial ET-1 and eNOS expression in intact human conduit vessels exposed to different intraluminal pressure levels at physiological shear stress

OBJECTIVE By using a computerized vascular perfusion model, we investigated temporal effects of sub-acute pressure elevation on vasomotor behavior and expression of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) in intact human conduit vessels. METHODS Paired umbilical veins were perfused during 1.5, 3 and 6 h under high/low intraluminal pressure (40/20 mmHg) and at identical shear stress level of 10 dyn/cm(2). ET-1 and eNOS gene and protein expression was quantified with real-time reverse-transcribed polymerase chain reaction and quantitative immunohistochemistry, respectively. RESULTS Pressure induced differential temporal regulation patterns of ET-1 and eNOS gene expression. During the high pressure condition, eNOS mRNA was upregulated after 3 h and leveled off after 6 h of perfusion, while ET-1 mRNA was elevated after 6 h perfusion. Immunohistochemistry verified synchronal changes at the protein level. Significant vasodilation was observed after 3 h in the high-pressure system. CONCLUSION Thus, subacute pressure elevation exerts differential effects on the endothelial eNOS/ET-1 expression, which dynamically regulate the vasomotor tone.

Differential immediate-early gene responses to shear stress and intraluminal pressure in intact human conduit vessels

We have previously shown distinct effects of shear stress and pressure on transcription of several potent vascular mediators. In the present study, we tested the hypothesis that c‐jun and c‐fos are regulated differentially by shear and pressure. Intact human umbilical veins were perfused with various combinations of shear and pressure during 1.5, 3 and 6 h. Protein and gene expressions were assessed by immunofluorescence and real‐time reverse transcription PCR, respectively. Shear stress and pressure exert differential temporal effects on c‐jun and c‐fos gene and protein expression, and these immediate‐early gene responses appear to be cell‐type specific for endothelial and smooth muscle cells.

Intraluminal pressure modulates eicosanoid enzyme expression in vascular endothelium of intact human conduit vessels at physiological levels of shear stress.

Objective Biosynthesis of eicosanoid metabolites in blood vessels regulates vascular tone and platelet function. We investigated whether intraluminal pressure modulates gene and protein expression of key eicosanoid enzymes in intact human conduit vessels and/or release of their vasoactive metabolites. Methods Paired segments of human umbilical veins were perfused under laminar flow for 1.5, 3 and 6 h at high versus low intraluminal pressure (40/20 mmHg) with identical shear stress (10 dyn/cm2). Endothelial cell mRNAs encoding cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostaglandin synthase (PGS), and thromboxane synthase (TXS) were measured by quantitative real-time RT-PCR. Secretion of PGI2 and TXA2 to the perfusion medium was measured by enzyme immunoassay of their metabolites 6-keto-prostaglandin F1β and TXB2. Results Intraluminal pressures were 39.9 ± 0.02 and 20.0 ± 0.03 mmHg (P < 0.0001) in high and low pressure circuits, and shear stress levels were 10.6 ± 0.60 and 9.7 ± 0.36 dyn/cm2 (NS, not significant). COX-1 mRNA was significantly up-regulated after 1.5 h of high pressure stimulation and continued up to 3 h, but fell thereafter significantly below baseline after 6 h. COX-2 mRNA was initially significantly down-regulated, followed by a significant up-regulation after 6 h. Gene expressions of PGS and TXS were significantly induced after 6 h of high pressure perfusion. High pressure depressed the production of PGI2 (P < 0.05) but did not alter TXA2 formation. Conclusions Intraluminal pressure has differential effects on gene and protein expression of key eicosanoid enzymes and biosynthesis of prostanoid metabolites in intact human conduit vessles. The new, computerized biomechanical perfusion system may be a useful tool to elucidate specific effects of various biomechanical forces on intact mammalian conduit vessels.