Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 µm) and density (0.045 vs. 0.57 µm^–2) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 µm^–3), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 µm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.

The vascular smooth muscle cell is a highly specialized cell whose primary function is contraction and relaxation. It expresses a variety of contractile proteins, ion channels, and signalling molecules that regulate contraction. Upon contraction, vascular smooth muscle cells shorten, thereby decreasing the diameter of a blood vessel to regulate the blood flow and pressure. Contractile activity in vascular smooth muscle cells is initiated by a Ca(2+)-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca(2+)-sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, thereby maintaining force. Removal of Ca(2+) from the cytosol and stimulation of myoson phosphatase initiate the relaxation of vascular smooth muscle.

Spontaneous tone in large arteries may contribute to the pathogenesis of hypertension. Reactive oxygen species and Ca 2ϩ influx have been shown to stimulate the development of spontaneous tone in isolated aortic rings in several models of hypertensive rats. The aim of this study was to investigate the role of the RhoA/Rho-kinase signaling pathway in the development of spontaneous tone in angiotensin II-induced hypertension and to explore the underlying mechanisms of RhoA/Rho-kinase activation. Our results showed that spontaneous tone was greatly enhanced in endothelium-denuded aortic rings from angiotensin II-induced hypertensive rats compared with their normotensive counterparts (73 Ϯ 5 versus 7 Ϯ 3% of phenylephrine-induced maximal contraction, respectively). The Rhokinase inhibitor (R)-(ϩ)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) (0.1-10 M) concentration dependently inhibited spontaneous tone in aortic rings from angiotensin II-treated rats. NADPH oxidase inhibitors diphenylene iodonium and apocynin also significantly reduced spontaneous tone. Chronic angiotensin II treatment markedly increased RhoA protein expression (57%) but had no effect on Rho guanine nucleotide exchange factor mRNA or Rho-kinase protein expression levels. In endothelium-denuded rings from normotensive rats, angiotensin II (100 nM) increased RhoA membrane translocation and phosphorylation of the myosin light chain phosphatase target subunit, which were both blocked by the NADPH oxidase inhibitor diphenylene iodonium (10 M). In conclusion, these data suggest that chronic treatment with angiotensin II leads to up-regulation of the RhoA/ Rho-kinase pathway, contributing to spontaneous tone development in rat aorta. Increased NADPH oxidase-dependent reactive oxygen species may be one of the mechanisms mediating the RhoA/Rho-kinase activation.Angiotensin II (Ang II) produced by the renin-angiotensin system is an important hormone in the homeostasis of cardiovascular and renal function. Increased circulating Ang II promotes inflammation and cell growth and increases vascular reactivity (Kagiyama et al., 2002;Seshiah et al., 2002). It is well known that activation of Ang II type I receptor (AT 1 ) increases free intracellular Ca 2ϩ concentration and myosin light chain (MLC) kinase activity, leading to MLC phosphorylation and subsequent smooth muscle contraction. Recent studies suggest that the phosphorylation state of MLC is also modulated by RhoA/Rho-kinase, a Ca 2ϩ sensitization signaling pathway through which smooth muscle is able to contract when the intracellular Ca 2ϩ concentration is low (Somlyo and Somlyo, 2003).The activity of RhoA is regulated by Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GDP disassociation inhibitors (Somlyo and Somlyo, 2003). In its inactive state, RhoA binds with GDP and forms a complex with GDP disassociation inhibitors in the cytosol. When RhoGEF is activated by G protein-coupled receptors such as AT 1 or receptor tyrosine kinase, it facilitates the exchange of ...

Ca+ -activated K+ -channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with Nomega-nitro-L-arginine methyl ester (L-NAME, 100 micromol/l) and indomethacin (10 micromol/l), and precontracted with phenylephrine (10 micromol/l). Cumulative concentration-response curves to ACh (0.001-10 micromol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBKalpha1 and rBKbeta1) genes were similar in both MA1 and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.

Ca(2+)-activated K(+) channels (K(Ca)), in particular, the small and intermediate K(Ca) (SK(Ca) and IK(Ca), respectively) channels, are key players in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SK(Ca) and IK(Ca) channels in ACh-induced relaxations is reduced due to decreased expression of SK(Ca) and IK(Ca) channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SK(Ca) channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of 1-day ANG II-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The IK(Ca) channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity.

Acute increases in cellular protein O-linked N-acetyl-glucosamine (O-GlcNAc) modification (O-GlcNAcylation) have been shown to have protective effects in the heart and vasculature. We hypothesized that d-glucosamine (d-GlcN) and Thiamet-G, two agents that increase protein O-GlcNAcylation via different mechanisms, inhibit TNF-α-induced oxidative stress and vascular dysfunction by suppressing inducible nitric oxide (NO) synthase (iNOS) expression. Rat aortic rings were incubated for 3h at 37°C with d-GlcN or its osmotic control l-glucose (l-Glc) or with Thiamet-G or its vehicle control (H(2)O) followed by the addition of TNF-α or vehicle (H(2)O) for 21 h. After incubation, rings were mounted in a myograph to assess arterial reactivity. Twenty-four hours of incubation of aortic rings with TNF-α resulted in 1) a hypocontractility to 60 mM K(+) solution and phenylephrine, 2) blunted endothelium-dependent relaxation responses to ACh and substance P, and 3) unaltered relaxing response to the Ca(2+) ionophore A-23187 and the NO donor sodium nitroprusside compared with aortic rings cultured in the absence of TNF-α. d-GlcN and Thiamet-G pretreatment suppressed the TNF-α-induced hypocontractility and endothelial dysfunction. Total protein O-GlcNAc levels were significantly higher in aortic segments treated with d-GlcN or Thiamet-G compared with controls. Expression of iNOS protein was increased in TNF-α-treated rings, and this was attenuated by pretreatment with either d-GlcN or Thiamet-G. Dense immunostaining for nitrotyrosylated proteins was detected in the endothelium and media of the aortic wall, suggesting enhanced peroxynitrite production by iNOS. These findings demonstrate that acute increases in protein O-GlcNAcylation prevent TNF-α-induced vascular dysfunction, at least in part, via suppression of iNOS expression.

Abstract-The vascular kallikrein-kinin system contributes to about one third of flow-dependent dilation in mice carotid arteries, by activating bradykinin B 2 receptors coupled to endothelial nitric oxide (NO) release. Because the bradykinin/NO pathway may mediate some of the effects of angiotensin II AT 2 receptors, we examined the possible contribution of AT 2 receptors to the kinin-dependent response to flow. Changes in outer diameter after increases in flow rate were evaluated in perfused arteries from wild-type animals (TK ϩ/ϩ ) and in tissue kallikrein-deficient mice (TK Ϫ/Ϫ ) in which the presence of AT 2 receptor expression was verified. Saralasin, a nonselective angiotensin II receptor antagonist, impaired significantly flow-induced dilation in TK ϩ/ϩ , whereas it had no effect in TK Ϫ/Ϫ mice. In both groups, blockade of AT 1 receptors with losartan or candesartan did not affect the response to flow. Inhibition of AT 2 receptors with PD123319 reduced significantly flow-induced dilation in TK ϩ/ϩ mice, but had no significant effect in TK Ϫ/Ϫ mice. Combining PD123319 with the bradykinin B 2 receptor antagonist HOE-140 had no additional effect to AT 2 receptor blockade alone in TK ϩ/ϩ arteries. Flow-dependent-dilation was also impaired in AT 2 receptor deficient mice (AT 2 Ϫ/Ϫ ) when compared with wild-type littermates. Furthermore, HOE-140 significantly reduced the response to flow in the AT 2 ϩ/ϩ , but not in AT 2 Ϫ/Ϫ mice. In conclusion, this study demonstrates that the presence of functional AT 2 receptors is necessary to observe the contribution of the vascular kinin-kallikrein system to flow-dependent dilation.

Objective-Tissue kallikrein (TK) participates in acute flow-induced dilatation (FID) of large arteries. We investigated whether TK deficiency blunts FID and alters chronic flow-related arterial structural and functional changes in resistance-sized muscular arteries. Methods and Results-Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, 18-to 19-day pregnant, and 7-day postpartum TK Ϫ/Ϫ and TK ϩ/ϩ littermate mice. In TK Ϫ/Ϫ mice, values of diameter, medial cross-sectional area (CSA), myogenic tone, and dilatation in response to acetylcholine were comparable to those values in TK ϩ/ϩ mice, but FID (0 to 100 L/min) was significantly reduced (55Ϯ4% versus 85Ϯ4% in TK ϩ/ϩ mice). In both mouse strains, pregnancy resulted in significant increases in diameter and medial CSA and in the N w -nitro-L-arginine methyl ester-sensitive component of FID. By 7 days after pregnancy, uterine arterial diameter and CSA values no longer differed from nonpregnant values, and FID was markedly reduced in TK Ϫ/Ϫ and TK ϩ/ϩ mice. Conclusions-These observations (1) confirm at the level of resistance arteries the key role of TK in FID and (2) Pressure and flow are hemodynamic determinants of circumferential wall stress and wall shear stress (WSS) in the arterial system. On an acute basis, they modulate arterial smooth muscle tone: an increase in transmural pressure triggers a myogenic contractile response, 6 -8 whereas an increase in flow induces an endothelium-dependent FID. 8 -11 Long-term changes in blood flow lead to arterial structural adaptations to normalize WSS. [12][13][14][15][16][17] In general, chronic blood flow elevations lead to a widening of lumen diameter, whereas sustained reductions in blood flow lead to a narrowing of lumen diameter. These structural arterial adaptations have been shown to be endothelium dependent. 18 Pregnancy may be considered a model of physiological flow-related remodeling of uterine arteries. It has previously been studied in rats, 19 guinea pigs, 20 and ewes. 21 During pregnancy, blood flow through the uterine circulation increases substantially. 22,23 To accommodate this increase in uterine blood flow, the uterine vasculature undergoes luminal expansion and an increase in wall mass, 19 a process termed outward hypertrophic remodeling or arteriogenesis. The driving force for this remodeling is increased WSS. Previous studies have demonstrated that chronic elevations in blood flow lead to an upregulation of FID, which is mainly due to an enhanced production of endothelial factors, such as NO and prostaglandins.The purpose of the present study was to investigate whether TK deficiency alters FID and chronic flow-related arterial structural and functional alterations in resistance-sized muscular arteries. Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, latepregnant, and postpartum TK Ϫ/Ϫ and TK ϩ/ϩ mice. Methods Mice Lacking TKTK-null mice were obtained by targeted disruption of the...