Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of α and β transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. β2 integrin (CD18) associates with four different α (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that β2 integrin is also expressed by other types of cells. Since the gene for β2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that β2 integrin and the αL, αM, and αX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of β2 integrin or against its α subunit partners, showed that β2 integrin, as well as the αL, αM, and αX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of β2 integrin in these various locations in the embryonic heart. These results indicate that the β2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart

Angiotensin-(1-7) activates a tyrosine phosphatase and inhibits glucose-induced signalling in proximal tubular cells

<b>Background.</b> In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1–7) [Ang-(1–7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1–7) on signalling responses in LLC-PK1 cells in normal (5 mM) or high (25 mM) glucose.<p></p> <b>Methods.</b> The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [3H]-leucine incorporation and transforming growth factor-β1 (TGF-β1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA.<p></p> <b>Results.</b> High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1–7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1–7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1–7) on high glucose-stimulated p38 MAPK. Ang-(1–7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-β1. Conversely, Ang-(1–7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV.<p></p> <b>Conclusions.</b> These data indicate that in proximal tubular cells, binding of Ang-(1–7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1–7) selectively inhibits glucose-stimulated protein synthesis and TGF-β1. In diabetic nephropathy, Ang-(1–7) may partly counteract the profibrotic effects of high glucose

Single-walled carbon nanotubes functionalized with sodium hyaluronate enhance bone mineralization

The aim of this study was to evaluate the effects of sodium hyaluronate (HY), single-walled carbon nanotubes (SWCNTs) and HY-functionalized SWCNTs (HY-SWCNTs) on the behavior of primary osteoblasts, as well as to investigate the deposition of inorganic crystals on titanium surfaces coated with these biocomposites. Primary osteoblasts were obtained from the calvarial bones of male newborn Wistar rats (5 rats for each cell extraction). We assessed cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay and by double-staining with propidium iodide and Hoechst. We also assessed the formation of mineralized bone nodules by von Kossa staining, the mRNA expression of bone repair proteins, and the deposition of inorganic crystals on titanium surfaces coated with HY, SWCNTs, or HY-SWCNTs. The results showed that treatment with these biocomposites did not alter the viability of primary osteoblasts. Furthermore, deposition of mineralized bone nodules was significantly increased by cells treated with HY and HY-SWCNTs. This can be partly explained by an increase in the mRNA expression of type I and III collagen, osteocalcin, and bone morphogenetic proteins 2 and 4. Additionally, the titanium surface treated with HY-SWCNTs showed a significant increase in the deposition of inorganic crystals. Thus, our data indicate that HY, SWCNTs, and HY-SWCNTs are potentially useful for the development of new strategies for bone tissue engineering

Impairment of in vitro and in vivo heart function in angiotensin-(1-7) receptor Mas knockout mice

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4±0.09 versus 2.1±0.03 g in Mas mice), ±dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart

Attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-(1-7)-producing fusion protein in the heart

OBJECTIVE: It has been shown that Ang-(1-7) has cardioprotective actions. To directly investigate the effects of Ang-(1-7) specifically in the heart, we generated and characterized transgenic (TG) rats which express an Ang-(1-7)-producing fusion protein driven by the alpha-MHC promoter. METHODS: and RESULTS: After microinjection of the transgene into fertilized rat zygotes, we obtained four different transgenic lines. Homozygous animals were analyzed with regard to the expression profile of the transgene by ribonuclease protection assay. Transgene expression was detected mainly in the heart with weak or no expression in other organs. Heterozygous TG(hA-1-7)L7301 rats presented a significant increase in cardiac Ang-(1-7) concentration compared with control rats (17.1 +/- 2.1 versus 3.9 +/- 1.4 pg/mg protein in SD rats). Radiotelemetry analysis revealed that TG rats presented no significant changes in blood pressure and heart rate compared with normal rats. Overexpression of Ang-(1-7) in the heart produced slight improvement in resting cardiac function (+ dT/dt: 81530 +/- 1305.0 versus 77470 +/- 345.5 g/s bpm in SD rats, p < 0.05), which was in keeping with the enhanced [Ca(2+)] handling observed in cardiomyocytes of TG rats. TG(hA-1-7)L7301 rats also showed a greater capacity to withstand stress since TG rats showed a less pronounced deposition of collagen type III and fibronectin induced by isoproterenol treatment in the subendocardial area than in corresponding controls. In addition, hearts from TG rats showed reduced incidence and duration of reperfusion arrhythmias in comparison with SD rats. CONCLUSION: These results indicate that Ang-(1-7) has blood pressure-independent, antifibrotic effects, acting directly in the heart

Genetic deletion of the angiotensin-(1-7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria

Angiotensin-(1-7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1-7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-beta mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney