Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Elastin organization in pig and cardiovascular disease patients' pericardial resistance arteries.

Peripheral vascular resistance is increased in essential hypertension. This involves structural changes of resistance arteries and stiffening of the arterial wall, including remodeling of the extracellular matrix. We hypothesized that biopsies of the human parietal pericardium, obtained during coronary artery bypass grafting or cardiac valve replacement surgeries, can serve as a source of resistance arteries for structural research in cardiovascular disease patients. We applied two-photon excitation fluorescence microscopy to study the parietal pericardium and isolated pericardial resistance arteries with a focus on the collagen and elastin components of the extracellular matrix. Initial findings in pig tissue were confirmed in patient biopsies. The microarchitecture of the internal elastic lamina in both the pig and patient pericardial resistance arteries (studied at a transmural pressure of 100 mm Hg) is fiber like, and no prominent external elastic lamina could be observed. This microarchitecture is very different from that in rat mesenteric arteries frequently used for resistance artery research. In conclusion, we add three-dimensional information on the structure of the extracellular matrix in resistance arteries from cardiovascular disease patients and propose further use of patient pericardial resistance arteries for studies of the human microvasculature.

The angiotensin AT2-receptor agonist compound 21 is an antagonist for the thromboxane TP-receptor - Implications for preclinical studies and future clinical use.

Since the AT2-receptor (AT2R) agonist C21 has structural similarity to the AT1-receptor antagonists Irbesartan and Losartan, which are antagonists not only at the AT1R, but also at thromboxane TP-receptors, we tested the hypothesis that C21 has TP-receptor antagonistic properties as well. Isolated mouse mesenteric arteries from C57BL/6 J and AT2R-knockout mice (AT2R-/y) were mounted in wire myographs, contracted with either phenylephrine or the thromboxane A2 (TXA2) analogue U46619, and the relaxing effect of C21 (0.1 nM - 10 µM) was investigated. The effect of C21 on U46619-induced platelet aggregation was measured by an impedance aggregometer. Direct interaction of C21 with TP-receptors was determined by an ß-arrestin biosensor assay. C21 caused significant, concentration-dependent relaxations in phenylephrine- and U46619-contracted mesenteric arteries from C57BL/6 J mice. The relaxing effect of C21 was absent in phenylephrine-contracted arteries from AT2R-/y mice, whereas it was unchanged in U46619-contracted arteries from AT2R-/y mice. C21 inhibited U46619-stimulated aggregation of human platelets, which was not inhibited by the AT2R-antagonist PD123319. C21 reduced U46619-induced recruitment of ß-arrestin to human thromboxane TP-receptors with a calculated Ki of 3.74 µM. We conclude that in addition to AT2R-agonistic properties, C21 also acts as low-affinity TP-receptor antagonist, and that - depending on the constrictor - both mechanisms can be responsible for C21-induced vasorelaxation. Furthermore, by acting as a TP-receptor antagonist, C21 inhibits platelet aggregation. These findings are important for understanding potential off-target effects of C21 in the preclinical and clinical context and for the interpretation of C21-related myography data in assays with TXA2-analogues as constrictor.

Relaxing Responses to Hydrogen Peroxide and Nitric Oxide in Human Pericardial Resistance Arteries Stimulated with Endothelin-1.

In human pericardial resistance arteries, effects of the endothelium-dependent vasodilator bradykinin are mediated by NO during contraction induced by K+ or the TxA2 analogue U46619 and by H2 O2 during contraction by endothelin-1 (ET-1), respectively. We tested the hypotheses that ET-1 reduces relaxing effects of NO and increases those of H2 O2 in resistance artery smooth muscle of patients with cardiovascular disease. Arterial segments, dissected from the parietal pericardium of 39 cardiothoracic surgery patients, were studied by myography during amplitude-matched contractions induced by K+ , the TXA2 analogue U46619 or ET-1. Effects of the NO donor Na-nitroprusside (SNP) and of exogenous H2 O2 were recorded in the absence and presence of inhibitors of cyclooxygenases, NO synthases and small and intermediate conductance calcium-activated K+ channels. During contractions induced by either of the three stimuli, the potency of SNP did not differ and was not modified by the inhibitors. In vessels contracted with ET-1, the potency of H2 O2 was on average and in terms of interindividual variability considerably larger than in K+ -contracted vessels. Both differences were not statistically significant in the presence of inhibitors of mechanisms of endothelium-dependent vasodilatation. In resistance arteries from patients with cardiovascular disease, ET-1 does not selectively modify smooth muscle relaxing responses to NO or H2 O2 . Furthermore, the candidate endothelium-derived relaxing factor H2 O2 also acts as an endothelium-dependent vasodilator.

Endothelin-1 shifts the mediator of bradykinin-induced relaxation from NO to H2 O2 in resistance arteries from patients with cardiovascular disease.

BACKGROUND AND PURPOSE: We tested the hypothesis that in resistance arteries from cardiovascular disease (CVD) patients, effects of an endothelium-dependent vasodilator depend on the contractile stimulus. EXPERIMENTAL APPROACH: Arteries dissected from parietal pericardium of cardiothoracic surgery patients were studied by myography and imaging techniques. Segments were sub-maximally contracted by K(+) , the TxA2 analogue U46619 or endothelin-1 (ET-1). KEY RESULTS: Relaxing effects of Na-nitroprusside were comparable, but those of bradykinin (BK) were bigger in the presence of ET-1 compared with K(+) or U46619. BK-induced relaxation was (i) abolished by L-NAME in K(+) -contracted arteries, (ii) partly inhibited by L-NAME in the presence of U46619 and (iii) not altered by indomethacin, L-NAME plus inhibitors of small and intermediate conductance calcium-activated K(+) channels, but attenuated by catalase, in ET-1-contracted arteries. This catalase-sensitive relaxation was unaffected by inhibitors of NADPH oxidases or allopurinol. Exogenous H2 O2 caused a larger relaxation of ET-1-induced contractions than those evoked by K(+) or U46619 in the presence of inhibitors of other endothelium-derived relaxing factors. Catalase-sensitive staining of cellular ROS with CellROX Deep Red was significantly increased in the presence of both 1 µM BK and 2 nM ET-1 but not either peptide alone. CONCLUSIONS AND IMPLICATIONS: In resistance arteries from patients with CVD, exogenous ET-1 shifts the mediator of relaxing responses to the endothelium-dependent vasodilator BK from NO to H2 O2 and neither NADPH oxidases, xanthine oxidase nor NOS appear to be involved in this effect. This might have consequences for endothelial dysfunction in conditions where intra-arterial levels of ET-1 are enhanced.

Early impairment of skeletal muscle endothelial glycocalyx barrier properties in diet-induced obesity in mice.

While previous studies have indicated an important role for the endothelial glycocalyx in regulation of microvascular function, it was recently shown that acute enzymatic glycocalyx degradation in rats was associated with an impaired insulin-mediated glucose disposal. The aim of this study was to determine whether glycocalyx damage in skeletal muscle occurs at an early stage of diet-induced obesity (DIO). The microcirculation of the hindlimb muscle of anesthetized C57Bl/6 mice, fed chow (CON) or a high-fat diet (HFD) for 6 and 18 weeks (w), respectively, was visualized with a Sidestream Dark-Field camera, and glycocalyx barrier properties were derived from the calculated perfused boundary region (PBR). Subsequently, an intraperitoneal glucose tolerance test was performed and the area under the curve (AUC) of blood glucose was calculated. Impairment of glycocalyx barrier properties was already apparent after 6 weeks of HFD and remained after 18 weeks of HFD (PBR [in µm]: 0.81 ± 0.03 in CON_6w vs. 0.97 ± 0.04 in HFD_6w and 1.02 ± 0.07 in HFD_18w [both P < 0.05]). Glucose intolerance appeared to develop more slowly (AUC [in mmol/L × 120 min]: 989 ± 61 in CON_6w vs. 1204 ± 89 in HFD_6w [P = 0.11] and 1468 ± 84 in HFD_18w [P < 0.05]) than the impairment of glycocalyx barrier properties. The data indicate that damage to the endothelial glycocalyx is an early event in DIO. It is suggested that glycocalyx damage may contribute to the development of insulin resistance in obesity.