Martin Paul

The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells.

Angiotensin II (ANG II) is known to be a potent growth promoting factor for vascular smooth muscle cells and fibroblasts but little is known about its influence on growth in endothelial cells. We studied the effects of ANG II on endothelial growth and the role of the angiotensin receptor subtypes involved. Proliferation of rat coronary endothelial cells (CEC) and rat vascular smooth muscle cells (VSMC) was determined by [3H]thymidine incorporation, the MTT-test and by directly counting cells in a coulter counter. Angiotensin AT1- and AT2-receptors were demonstrated by binding studies and by the presence of their respective mRNA through reverse transcription polymerase chain reaction (RT-PCR). In contrast to VSMC, which in culture only express the AT1-receptor, CEC express both, AT1- and AT2-receptors simultaneously up to the third passage. Whereas ANG II stimulated growth of quiescent VSMC, an effect abolished by pretreatment with the AT1-receptor antagonist, losartan, ANG II did not induce proliferation in quiescent CEC. However, after pretreatment of quiescent endothelial cells (< passage 4) with the AT2-receptor antagonist, PD 123177, ANG II induced proliferation. This effect was reversed by additional pretreatment with losartan. ANG II significantly inhibited the proliferation of bFGF-stimulated CEC in a dose-dependent manner by maximally 50%. This effect was prevented by PD 123177 while losartan was ineffective. The AT2-receptor agonist, CGP 42112, mimicked the antiproliferative actions of ANG II, confirming the specificity of the effect. Our results show that the growth modulating actions of ANG II depend on the type of angiotensin receptor present on a given cell. In coronary endothelial cells, the antiproliferative actions of the AT2-receptor offset the growth promoting effects mediated by the AT1-receptor.

Tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure.

In addition to the circulating renin-angiotensin system, recent data demonstrate the existence of tissue renin-angiotensin systems that may be important in cardiovascular homeostasis. However, the relative activities of the circulating and tissue renin-angiotensin systems have not been examined previously in pathophysiological states, such as congestive heart failure. The present study was performed to examine the status of plasma and tissue angiotensin converting enzyme (ACE) activities in compensated experimental heart failure induced by coronary artery ligation in the rat. Three groups of male Sprague-Dawley rats were examined: 1) nonoperated rats (NO, n = 5), 2) sham-operated rats (SO, n = 5), and 3) heart failure rats (HF, n = 11). Rats were studied an averaged of 85 days postoperatively. In HF animals, plasma renin concentration and serum ACE activities were not different compared with NO and SO control animals. Cardiac ACE activity was 50% greater in the right ventricle than the interventricular septum in NO and SO rats. Both right ventricular and interventricular septal ACE activity increased approximately twofold in HF animals as compared with NO and SO groups (p less than 0.05). In contrast, pulmonary, aortic, and renal ACE activities were not altered in HF rats compared with control animals. A positive correlation existed between the histopathological size of myocardial infarction and the level of right ventricular ACE activity (r = 0.75, p less than or equal to 0.05). Such a relation between infarct size and either serum or noncardiac tissue ACE activities was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.

Blockade of the Intermediate-Conductance Calcium-Activated Potassium Channel as a New Therapeutic Strategy for Restenosis

Background—Angioplasty stimulates proliferation and migration of vascular smooth muscle cells (VSMC), leading to neointimal thickening and vascular restenosis. In a rat model of balloon catheter injury (BCI), we investigated whether alterations in expression of Ca2+-activated K+ channels (KCa) contribute to intimal hyperplasia and vascular restenosis. Methods and Results—Function and expression of KCa in mature medial and neointimal VSMC were characterized in situ by combined single-cell RT-PCR and patch-clamp analysis. Mature medial VSMC exclusively expressed large-conductance KCa (BKCa) channels. Two weeks after BCI, expression of BKCa was significantly reduced in neointimal VSMC, whereas expression of intermediate-conductance KCa (IKCa1) channels was upregulated. In the aortic VSMC cell line, A7r5 epidermal growth factor (EGF) induced IKCa1 upregulation and EGF-stimulated proliferation was suppressed by the selective IKCa1 blocker TRAM-34. Daily in vivo administration of TRAM-34 to rats significantly reduced intimal hyperplasia by ≈40% at 1, 2, and 6 weeks after BCI. Two weeks of treatment with the related compound clotrimazole was equally effective. Reduction of intimal hyperplasia was accompanied by decreased neointimal cell content, with no change in the rate of apoptosis or collagen content. Conclusions—The switch toward IKCa1 expression may promote excessive neointimal VSMC proliferation. Blockade of IKCa1 could therefore represent a new therapeutic strategy to prevent restenosis after angioplasty.

Gene expression of the renin-angiotensin system in human tissues. Quantitative analysis by the polymerase chain reaction.

Activation of tissue-specific gene expression of the components of the renin-angiotensin system (RAS) in humans may play an important role in cardiovascular regulation and pathophysiology. Studies of human tissue RAS expression, however, have been limited by the lack of availability of sufficient amounts of fresh human tissues and a sensitive method for detecting specific mRNAs. To demonstrate the presence of components of local RASs in humans we used the polymerase chain reaction (PCR) after reverse transcription to detect renin- angiotensinogen-, and angiotensin-converting enzyme-mRNA in small quantities of human tissues. Results indicated that all components of the RAS were widely expressed in human organ samples. In order to study changes of gene expression in small tissue samples (e.g., renal biopsies) obtained from patients, we established a competitive PCR assay for quantification of renin, using a 155-basepair deletion mutant of the human renin cDNA as an internal standard. Renin-mRNA concentration was quantitated in the kidney (1.74 +/- 0.2 pg renin/micrograms total RNA), adrenal gland (1.15 +/- 0.15 pg renin/micrograms total RNA), placenta (0.7 +/- 0.1 pg renin/micrograms total RNA), and saphenous vein (0.02 +/- 0.01 pg renin/micrograms total RNA). The method described here may serve as a highly sensitive tool to quantify alterations in gene expression in man under various pathophysiologic conditions. This study should provide the methodological basis for future studies of tissue RAS in human physiology and disease.

Oxidative stress increases endothelin-1 synthesis in human coronary artery smooth muscle cells.

&NA; Endothelins, nitric oxide, and oxygen‐derived free radicals decisively regulate vascular tone. An imbalance in the biosynthesis of these substances in pathophysiologic conditions may trigger vasospasm and promote the development of atherosclerosis. Previous studies have shown that oxygen‐derived free radicals can increase the synthesis of endothelin‐1 in cultured endothelial cells. Interestingly, conditions of increased oxidative stress within smooth muscle cells as induced by angiotensin II infusion or hypercholesterolemia have been shown to be associated with increased autocrine synthesis of endothelin‐1. Because endothelin‐1 formed in smooth muscle cells can trigger hypersensitivity to vasoconstrictors, we tested whether oxidative stress per se may affect endothelin expression in vascular smooth muscle cells. Cultured human coronary artery smooth muscle cells were exposed to oxidative stress generated by the xanthine/xanthine oxidase reaction or by hydrogen peroxide. Preproendothelin‐1 mRNA content was quantitated by means of quantitative polymerase chain reaction and endothelin‐1 protein was measured by radioimmunoassay. Incubation with xanthine/xanthine oxidase significantly increased preproendothelin‐1 mRNA synthesis, whereas GAPDH remained unchanged. Likewise, xanthine/xanthine oxidase also led to a dose‐dependent increase of intracellular endothelin‐1. The increase in ET‐1 expression induced by xanthine/xanthine oxidase was significantly inhibited by superoxide dismutase but not by catalase. We conclude that oxygen‐derived free radicals can stimulate the synthesis of endothelin‐1 in endothelial and vascular smooth muscle cells by increasing preproendothelin‐1 mRNA content and that this effect is mediated predominantly by superoxide anions. We therefore have identified a new mechanism in the interaction of oxidative stress and endothelin‐1 expression in smooth muscle cells that may have important implications in diseases such as atherosclerosis and hypertension.

Expression and Function of Endothelial Ca2+-Activated K+ Channels in Human Mesenteric Artery A Single-Cell Reverse Transcriptase–Polymerase Chain Reaction and Electrophysiological Study In Situ

Ca2+-activated K+ (KCa) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase–polymerase chain reaction analysis in combination with the patch-clamp technique to characterize KCa channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial KCa channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non–cancer patients with inactive diverticulitis. Expression of the intermediate-conductance KCa channel (hIK1) was detected in non–cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding KCa currents, whereas respective KCa currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non–cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P <0.05). However, KCa current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance KCa channel (hSlo) was detected in single ECs from CA patients. The increased KCa channel expression in CA patients resulted in a 2.7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P <0.05). This increased expression and function of KCa channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Uridine adenosine tetraphosphate: a novel endothelium- derived vasoconstrictive factor

Beyond serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors (EDCFs) remains unclear. The supernatant obtained from mechanically stimulated human endothelial cells obtained from dermal vessels elicited a vasoconstrictive response in an isolated perfused rat kidney. A purinoceptor blocker had a greater effect than an endothelin receptor blocker in decreasing endothelially derived vasoconstriction in the isolated perfused rat kidney. The nucleotide uridine adenosine tetraphosphate (Up4A) was isolated from the supernatant of stimulated human endothelium and identified by mass spectrometry. Up4A is likely to exert vasoconstriction predominantly through P2X1 receptors, and probably also through P2Y2 and P2Y4 receptors. Plasma concentrations of Up4A that cause vasoconstriction are found in healthy subjects. Stimulation with adenosine 5′-triphosphate (ATP), uridine 5′-triphosphate (UTP), acetylcholine, endothelin, A23187 and mechanical stress releases Up4A from endothelium, suggesting that Up4A contributes to vascular autoregulation. To our knowledge, Up4A is the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. We conclude that Up4A is a novel potent nonpeptidic EDCF. Its vasoactive effects, plasma concentrations and its release upon endothelial stimulation strongly suggest that Up4A has a functional vasoregulatory role.

Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML‐1. Cultured on a three‐dimensional clinostat, ML‐1 cells formed three‐dimensional MCTSs (MCTS diameter: 0.3±0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up‐regulated the thyroid‐stimulating hormone‐receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML‐1 cells grown on the clinostat showed elevated amounts of the apoptosis‐associated Fas protein, of p53, and of bax but showed reduced quantities of bcl‐2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase‐mediated dUTP digoxigenin nick end labeling, 4′, 6‐diamidino‐2‐phenylindole staining, DNA laddering, and 85‐kDa apoptosis‐related cleavage fragments. These fragments resulted from enhanced 116‐kDa poly(ADP‐ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

A lack of genetic linkage of renin gene restriction fragment length polymorphisms with human hypertension.

&NA; Because renin is an important enzyme in blood pressure regulation, we studied the possibility that an alteration in the structure of the human renin gene is genetically linked to human essential hypertension or associated with levels of plasma renin activity or blood pressure. By using specific DNA probes, we have identified four polymorphisms in the human renin gene with the restriction enzymes Taq I, HindIII, Bgl I, and Bgl II. The gene location of all of these polymorphisms except for the Bgl II polymorphism has been determined, and their frequencies were initially estimated in a population of 50 random subjects. To test the clinical significance of these polymorphisms, we studied 68 persons from a large Utah pedigree with a high incidence of hypertension. Among nine relatives with hypertension, genetic linkage without recombination was ruled out by observing several obligate recombinants. We also found no significant association of the restriction fragment length polymorphisms with quantitative measurements of sitting or standing, systolic or diastolic blood pressures, or plasma renin activity in 59 untreated members of this pedigree. Although we found no genetic linkage in this set of study subjects, the characterization of the restriction fragment length polymorphisms for the renin gene may be useful in future studies of other selected pedigrees for the presence of one or more of these to be a genetic marker in hypertension. (Hypertension 1989;14:614‐618)