Arteries Of Hypertensive Animals Research Articles

O65 SARCOPLASMIC CALCIUM ATPASE (SERCA) PROTEOLYSIS BY MATRIX METALLOPROTEINASE (MMP)-2 CONTRIBUTES TO VASCULAR DYSFUNCTION IN EARLY HYPERTENSION

Background and objective: Hypertension-induced increased matrix metalloproteinase (MMP)-2 activity proteolyzes extra- and intracellular proteins in aortas and contributes to vascular dysfunction. MMP-2 proteolyzes sarcoplasmic reticulum calcium ATPase (SERCA) in the heart during ischemia and reperfusion injury, thus impairing cardiac function. SERCA activity is also impaired in the arteries of hypertensive animals. We investigated whether hypertension-induced increased MMP-2 activity in aorta contributes to SERCA proteolysis, vascular remodeling and dysfunction. Methods: Aortas from male Sprague-Dawley (SD) rats, incubated or not with angiotensin II (AngII) and MMP inhibitors, ONO-4817 or doxycycline (Doxy), were used for zymography and co-immunoprecipitation of SERCA with MMP-2. Male SD rats were also submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with Doxy from third to seventh day post-surgery. Systolic blood pressure (SBP) was assessed basally and daily by tail-cuff plethysmography. After 7 days, aortas were collected for gel and in situ zymography, western blot to SERCA, ATPase activity assay, vascular reactivity to phenylephrine and acetylcholine, Ki-67 immunofluorescence and hematoxylin/eosin stain. A7r5 cells were incubated or not with AngII, in the presence or absence of SERCA inhibitor, tapsigargin, to assess intracellular calcium concentrations. Results: AngII increased MMP-2 activity in aortas and MMP-2 co-immunoprecipitated with SERCA. SBP was increased in 2K-1C rats and Doxy did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Intracellular calcium concentrations were increased in A7r5 cells incubated with AngII and/or SERCA inhibitor, tapsigargin. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and Doxy decreased Ki-67 cell proliferation. In 2K-1C rats, arterial contraction to phenylephrine was accentuated but Doxy ameliorated only the impaired relaxation to acetylcholine. Conclusion: Doxy reduced MMP-2 activity in the aortas of 2K-1C rats and prevented the ATPase activity impairment and MMP-2-induced SERCA proteolysis and vascular dysfunction.

Retraction: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway.

Stiffening of large arteries is increasingly used as an independent predictor of risk and therapeutic outcome for small artery dysfunction in many diseases including pulmonary hypertension. The molecular mechanisms mediating downstream vascular cell responses to large artery stiffening remain unclear. We hypothesize that high pulsatility flow, induced by large artery stiffening, causes inflammatory responses in downstream pulmonary artery endothelial cells (PAECs) through toll-like receptor (TLR) pathways. To recapitulate the stiffening effect of large pulmonary arteries that occurs in pulmonary hypertension, ultrathin silicone tubes of variable mechanical stiffness were formulated and were placed in a flow circulatory system. These tubes modulated the simulated cardiac output into pulsatile flows with different pulsatility indices, 0.5 (normal) or 1.5 (high). PAECs placed downstream of the tubes were evaluated for their expression of proinflammatory molecules (ICAM-1, VCAM-1, E-selectin and MCP-1), TLR receptors and intracellular NF-κB following flow exposure. Results showed that compared to flow with normal pulsatility, high pulsatility flow induced proinflammatory responses in PAECs, enhanced TLR2 expression but not TLR4, and caused NF-κB activation. Pharmacologic (OxPAPC) and siRNA inhibition of TLR2 attenuated high pulsatility flow-induced pro-inflammatory responses and NF-κB activation in PAECs. We also observed that PAECs isolated from small pulmonary arteries of hypertensive animals exhibiting proximal vascular stiffening demonstrated a durable ex-vivo proinflammatory phenotype (increased TLR2, TLR4 and MCP-1 expression). Intralobar PAECs isolated from vessels of IPAH patients also showed increased TLR2. In conclusion, this study demonstrates for the first time that TLR2/NF-κB signaling mediates endothelial inflammation under high pulsatility flow caused by upstream stiffening, but the role of TLR4 in flow pulsatility-mediated endothelial mechanotransduction remains unclear.

Effect of 20-HETE inhibition on L-NAME-induced hypertension in rats

ABSTRACT20-Hydroxyeicosatetraenoicacid (20-HETE) is an important mediator that regulates vascular tone and blood pressure (BP). Although various experimental animal hypertension models demonstrated that 20-HETE contributes to increased vascular resistance and BP, these effects have not been studied in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension model. In this study, we investigated the effects of 20-HETE on the vascular responsiveness and BP in an L-NAME-induced hypertension.Wistar Albino rats were used in this study. Hypertension was induced by the addition of L-NAME to drinking water for 5 weeks. The study was performed in three stages: first, BP changes were monitored in real time in the presence of 20-HETE enzymatic inhibitor, N-hydroxy-N´-(4-butly-2-methylphenyl)-formamidine (HET-0016) for 1 h. Second, vascular responses of the conduit and resistance arteries were investigated in the presence or absence of HET-0016 in the organ bath. Third, BP was monitored weekly in some hypertensive animals treated with HET-0016 and vascular responses were investigated at the end of the experiment.We demonstrated an increase in 20-HETE levels in the resistance arteries of hypertensive animals. 20-HETE inhibition by HET-0016 significantly decreased BP in L-NAME-induced hypertension model. In addition, HET-0016 treatment caused significant improvement in vascular dilator and constrictor responses in the conduit and resistance arteries.This study demonstrates an important role of 20-HETE in increasing BP and altering vascular responsiveness in L-NAME-induced hypertension model, which suggests a possible involvement of 20-HETE in essential hypertension development in humans.

Genetic deletion of the TRPC3 channel blunts the development of angiotensin II‐induced hypertension in mice

The Transient Receptor Potential Channel 3 (TRPC3) is a cation – conducting channel in the plasma membrane of vascular smooth muscle cells, which is activated by G‐protein coupled receptors (GPCR) including the α1‐adrenergic receptor. Several reports indicate that TRPC3 are upregulated in arteries of hypertensive animals and humans, but the lack of a selective TRPC3 blocker has thwarted efforts to define its role in blood pressure elevation. Here, we used TRPC3−/− mice to define the contribution of TRPC3 to the pathogenesis of hypertension. TRPC3 mRNA level was similar between mesenteric arteries (MA) of Ang II‐ and saline‐infused WT mice, but TRPC3 protein expression was ~5.7‐fold higher in MA of hypertensive mice. Resting mean arterial pressure (MAP) was not different between TRPC3−/− and WT mice (119±3 vs. 125±3 mm Hg; n=8–10). However, Ang II infusion for 14 days increased MAP in WT mice to 170±3 mm Hg compared to only 146±4 mm Hg in TRPC3−/− mice. PE‐induced constrictions were blunted in pressurized MA of Ang II‐infused TRPC3−/− (53±2%) compared to WT mice (71±5%), and PE‐evoked cation current was lower in patch‐clamped VSMCs of Ang II‐infused TRPC3−/− mice (‐2.5±0.5 pA/pF) compared to WT animals (−5.4±0.6 pA/pF). Thus, TRPC3 appears to importantly contribute to Ang II‐induced hypertension possibly by mediating GPCR cation current in VSMCs. NIH R01 HL064806 (NJR), AHA 12PRE11850002 (ARP) and NIH Z01‐ES‐101684 (LB)

CC chemokine receptor 2 is required for macrophage infiltration and vascular hypertrophy in angiotensin II-induced hypertension.

Recent studies have identified the presence of macrophages in the arterial wall of hypertensive animals and suggested that as is the case in atherosclerosis, macrophage products may be important mediators of the adaptive response of the arterial wall. In support of this, we have previously shown that the expression of monocyte chemoattractant protein-1 is upregulated in the arteries of hypertensive animals. We hypothesized that macrophage recruitment is a critical step in the pathogenesis of hypertension. To obtain insights into this potential mechanism, we made use of mice deficient in the CC chemokine receptor 2 (CCR2), the receptor for monocyte chemoattractant protein-1. Hypertension was induced with the subcutaneous administration of angiotensin II (0.75 mg. kg(-1). d(-1)) for 7 days. Using in situ hybridization with a probe for c-fms to identify macrophages, we found that hypertension-induced macrophage infiltration of the arterial wall was virtually eliminated in CCR2-deficient mice. In addition, vascular hypertrophy was reduced by approximately 65% compared with wild-type animals. These data demonstrate that CCR2 is essential for the recruitment of macrophages into the arterial wall in the setting of hypertension. Furthermore, the decreased hypertrophic response suggests that vascular hypertrophy occurs in part as a consequence of macrophage infiltration. In angiotensin II-induced hypertension, CCR2-mediated responses are critical to the process of macrophage recruitment and vascular hypertrophy and may represent one mechanism by which at least some forms of hypertension may lead to the development of atherosclerosis.

Effect of Long-term Treatment With Propionyl- l -Carnitine on Smooth Muscle Cell Polyploidy in Spontaneously Hypertensive Rats

Experimental studies suggest that DNA content is increased in the smooth muscle cells of the arteries of hypertensive animals. It is unclear whether an increase in DNA content occurring in the smooth muscle cells of hypertensive rats represents a pressure-dependent effect. To evaluate the antihypertensive effect of long-term treatment with propionyl-L-carnitine and the possible morphological changes in thoracic smooth muscle cells correlated with this effect, we studied 4-month-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) randomly divided into five groups. One group of SHR was treated with propionyl-L-carnitine for 12 months; the other four groups of SHR and WKY received no treatment and were controls. We used static and flow cytometry to evaluate the polyploid cell content in thoracic aorta smooth muscle cells. Systolic pressure in untreated SHR progressively increased during the experiment. Treatment did not significantly influence pressure values in SHR. In WKY, blood pressure was significantly lower than that in treated and untreated age-matched SHR (2P < .02). The number of polyploid smooth muscle cells was significantly lower in the propionyl-L-carnitine-treated SHR than in the untreated rats (2P < .04) and similar to values for WKY. The reduction of polyploid cells in treated SHR was paralleled by a significant decrease of the aortic total DNA content, whereas no modifications occurred in smooth muscle cell mass. Long-term treatment with propionyl-L-carnitine may interfere with cellular mechanisms regulating the secondary responses involved in DNA synthesis.

Growth response of tetraploid smooth muscle cells to balloon embolectomy-induced vascular injury in the spontaneously hypertensive rat.

Previous studies in this laboratory have demonstrated that smooth muscle hypertrophy in large arteries of hypertensive animals is accompanied by development of polyploidy in a large fraction of the smooth muscle cell population. The present studies address whether there is an inherent loss in the ability of tetraploid cells to respond to proliferative stimuli by examining whether these cells initiate DNA replication after balloon catheter-induced vascular injury. Left carotid arteries of 5- to 7-month-old spontaneously hypertensive rats were subjected to balloon injury, and the animals were perfused intravenously with [3H]thymidine for 32 h. The animals were killed, and smooth muscle cells were isolated from carotid medial preparation by enzymatic digestion. Cells were processed for simultaneous autoradiographic analysis of [3H]thymidine incorporation and fluorometric analysis of cellular DNA content as a means of simultaneously assessing both the ploidy level of a cell and whether or not it had initiated DNA replication in response to balloon injury. Results demonstrated the presence of [3H]thymidine-labeled cells with DNA contents that were greater than 4C but less than 8C (p less than 0.01), indicating that they are cycling tetraploid cells. These data demonstrate that tetraploid smooth muscle cells in vivo are capable of initiating DNA synthesis in response to balloon injury. Results indicate that there is not an inherent loss in the capacity of tetraploid cells to respond to a proliferative stimulus, although it remains to be determined whether the polyploid cells that initiate DNA replication after balloon injury actually go on to divide.

Scanning electron microscopy of pulmonary vascular endothelium in rats with hypoxia-induced hypertension.

Scanning electron microscopy was used to study the endothelial surface of the pulmonary trunk, artery, and vein in normobaric control rats as well as in rats exposed to hypobaric hypoxia for 7 and 21 days. The individual endothelial cells of the normobaric pulmonary trunk and hilar artery were flat and slightly elongated with elevated nuclear regions, and those of the intermediate-sized artery were more elongated and had more microvilli than the large arteries studied. Their endothelial cell boundaries were outlined by beaded cytoplasmic projections. The surfaces of the normobaric hilar and intermediate-sized veins were smooth and demonstrated numerous longitudinal streaks. These venous endothelial cells were elongated and their cell boundaries were outlined by low discontinuous marginal folds. Exposure to hypobaric hypoxia caused the following changes on the arterial surface: elevation of the endothelial cells; formation of microvilli-rich cell clusters; formation of hollow defects; and the attachment of leukocytes. Hypobaric hypoxia also caused the disappearance of the longitudinal streaks and the occurrence of microvilli-rich cells in the hilar veins. The endothelial surface modifications in the hypobaric rats could be related to thickening of the endothelium, intimal edema, increased intimal connective tissue, luminal invasion of leukocytes, and increased endothelial cell proliferation, known to occur in systemic arteries of hypertensive animals.

The correlation between the development of sympathetic innervation and the development of medial hypertrophy in jejunal arteries in normotensive and spontaneously hypertensive rats

The catecholaminergic innervation of jejunal arteries was examined during development in normotensive and hypertensive rats. It was determined that a hyperinnervation of jejunal vessels in the hypertensive rats is present from 2 weeks of age onwards, before significant elevation of blood pressure and medial hypertrophy of jejunal arteries occurs. It is concluded that this hyperinnervation may be causally related to the medial hypertrophy in arteries of hypertensive animals.

Smooth muscle cell hypertrophy versus hyperplasia in hypertension.

Arteries of hypertensive animals have a greater mass of smooth muscle than those of normotensive controls. We examined the contribution of smooth muscle cell hypertrophy and hyperplasia to this increase in mass. Cell size measurements obtained by (i) image analysis of enzyme-dispersed cells, (ii) morphometric evaluation of tissue sections, and (iii) biochemical measures of protein/cell and actin/cell ratios on isolated cells showed that average cell size was greater in spontaneously hypertensive rats than in normotensive Wistar-Kyoto and Sprague-Dawley controls. Average DNA/cell ratios were also increased in spontaneously hypertensive rats while protein/DNA ratios were not different. Analysis of nuclear DNA content of individual cells by flow microfluorimetry and Feulgen-DNA microdensitometry measurements showed that greater than 20% of spontaneously hypertensive rats cells were polyploid while less than 10% of control cells were polyploid. Estimates of cell number per centimeter of aortic length, based on ploidy and DNA content, show no difference between control and hypertensive rats. Thus, smooth muscle hypertrophy alone accounts for the increased mass of smooth muscle in aortas of spontaneously hypertensive rats. Furthermore, this cellular hypertrophy is accompanied by a change in nuclear ploidy. This nuclear response in hypertension may represent a fixed change related to the establishment of a chronic hypertensive state.

Ionic composition of arterial wall in experimental hypertension.

1. To obtain additional information on the role of the ionic composition of the vascular wall in the pathogenesis of arterial hypertension, we studied the distribution of ions in aorta and tail arteries of rats with experimental renal hypertension. 2. The water content of rat aorta is markedly increased in hypertensive animals [1992 ml/kg-dry weight (DW) compared with 1688 ml/kg DW]. 3. Na, K, Ca, Mg and to a less extent Cl content is increased in both aorta and tail arteries. 4. These modifications are not affected by incubation in vitro. 5. Compartmental analysis as well as pharmacological studies suggest that part of the water increase and most of the potassium excess are related to a hypertrophy of smooth-muscle cells in hypertensive arteries. 6. The sodium excess in the vessels of hypertensive rats is bound in the extracellular space on anionic sites. 7. From these observations, it seems unlikely that the ionic modification described could affect the membrane potential of smooth-muscle cells, since the ratio between extra- and intra-cellular concentration is not markedly affected. 8. The bound sodium could affect the viscoelastic properties of the arteries of hypertensive animals. Cell hypertrophy by itself could explain at least partly the modification of the ionic composition and the increased reactivity of hypertensive vessels.