Sverker Jern

The vascular endothelium transduces physical stimuli within the circulation into physiological responses, which influence vascular remodelling and tissue homeostasis. Therefore, a new computerized biomechanical ex vivo perfusion system was developed, in which small intact vessels can be perfused under well-defined biomechanical forces. The system enables monitoring and regulation of vessel lumen diameter, shear stress, mean pressure, variable pulsatile pressure and flow profile, and diastolic reversal flow. Vessel lumen measuring technique is based on detection of the amount of flourescein over a vessel segment. A combination of flow resistances, on/off switches, and capacitances creates a wide range of pulsatile pressures and flow profiles. Accuracy of the diameter measurement was evaluated. The diameters of umbilical arteries were measured and compared with direct ultrasonographic measurement of the vessel diameter. As part of the validation the pulsatile pressure waveform was altered, e.g., in terms of pulse pressure, frequency, diastolic shape, and diastolic reversal flow. In a series of simulation experiments, the hemodynamic homeostasis functions of the system were successfully challenged by generating a wide range of vascular diameters in artificial and intact human vessels. We conclude that the system presented may serve as a methodological and technical platform when performing advanced hemodynamic stimulation protocols.

Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P = .001). Metabolic radiolabeling with ³⁵S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). This functional assay showed that the majority of the new protein was in an active configuration and could complexbind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots. (Blood. 2004;104:3943-3948)

Extracellular nucleotides such as ATP and UTP are released by activation of platelets and ischemic tissue injury. The aim of the present study was to investigate whether ATP and UTP can induce acute tPA release from the vascular endothelium in vivo. Nine healthy subjects were studied in a perfused-forearm model during stepwise intraarterial infusions of ATP and UTP (10-200 nmol/min), and UTP during inhibition of prostanoid and NO synthesis by indomethacin and L-NMMA. ATP and UTP induced a similar and marked stimulation of forearm tPA release which increased 11- and 18-fold above baseline (p ≤ 0.01 for both) in conjunction with pronounced vasodilation. Neither the acute tPA release nor the vasodilation could be abrogated by NO and prostanoid synthesis inhibition. The similar effect of ATP and UTP suggests that P2Y rather than adenosine receptors mediate the response. Release of extracellular nucleotides in ischemic tissue may induce a pronounced activation of the endogenous fibrinolytic system.

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/cm². 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.

We investigated the effect of shear stress on the expression of tissuetype plasminogen activator (t-PA) in intact human conduit vessels. Human umbilical veins were exposed to high or low shear stress (25 vs < 4 dyn/cm²) at identical intraluminal pressure (20 mmHg) for 1.5, 3, and 6 h in a new computerized biomechanical perfusion system. High shear perfusion induced a marked, time-dependent increase in t-PA immunostaining in both the endothelium and the media. t-PA relative to GAPDH gene expression increased by 54 ± 14% in highcompared to low-sheared vessels (p = 0.002). By contrast, t-PA release into the perfusion medium was similar in vessels perfused under high or low shear stress conditions. The results show that shear stress independently of pressure is a potent fluid mechanical stimulus for upregulation of the intracellular storage pool of t-PA in the vascular wall of fresh human conduit vessels. The shear effect is associated with an increased t-PA gene expression.