Yung S. Do

Angiotensin II Has Multiple Profibrotic Effects in Human Cardiac Fibroblasts

BACKGROUND Angiotensin II (Ang II) is implicated in cardiac remodeling and is increasingly recognized for its profibrotic activity. METHODS AND RESULTS Because little is known about the direct cellular effects of Ang II on human cardiac fibroblasts, we isolated fibroblasts from ventricles of explanted human hearts and added Ang II (100 nmol/L) to determine its role in growth, extracellular matrix accumulation, and adhesion. To assess which Ang II receptor is involved, Ang II was added in the presence of irbesartan (10 micromol/L), a specific AT(1) receptor antagonist; PD 123319 (10 micromol/L), a specific AT(2) receptor antagonist, or divalinil (100 nmol/L), an AT(4) receptor inhibitor. In human ventricles (n=13), message levels of atrial natriuretic peptide and AT(1) receptor were inversely correlated, which suggests a decrease in AT(1) receptor expression with progressive heart failure. Northern analysis and fluorescence-activated cell sorting demonstrated AT(1) receptor in cultured human cardiac fibroblasts. Ang II increased mitogen-activated protein kinase activity and in DNA synthesis (5-fold, P<0.01) stimulated a 2-fold increase in transforming growth factor-beta(1) (P<0.05) mRNA levels at 2 hours and a 2-fold increase in laminin (P<0.05) and fibronectin (P<0.05) mRNA levels at 24 hours. Ang II also enhanced plasminogen activator inhibitor-1 expression, which inhibits metalloproteinases that degrade the extracellular matrix. All of these effects were inhibited by irbesartan but not PD 123319 or divalinil. In addition, Ang II increased cardiac fibroblast attachment to collagens I and III, which was associated with an increase in focal adhesion kinase activity. CONCLUSIONS Activation of the AT(1) receptor in human heart promotes fibrosis. Ang II plays a novel role in stimulation of plasminogen activator inhibitor-1 expression and adhesion of cardiac fibroblasts to collagen.

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

Myocardial Osteopontin Expression Is Associated With Left Ventricular Hypertrophy

BACKGROUND Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes. METHODS AND RESULTS Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold (P<.01). OP mRNA was prominent in ventricular tissue from neonatal and adult rats with renovascular hypertension and aortic banding, whereas barely detectable levels were observed in normal adult cardiac tissue. ANP and OP mRNA levels in normal and hypertrophied ventricles correlated (r2=.87, P<.001). OP immunoreactivity and mRNA transcripts were predominantly found in cardiomyocytes not associated with inflammatory cells in sections from neonatal and adult hypertrophied hearts. No staining was detectable in normal adult hearts. Human myocardium with extensive fibrosis and cardiomyocyte hypertrophy obtained from explanted hearts with either idiopathic (n=5) or ischemic cardiomyopathy (n=7) demonstrated substantial myocyte immunoreactivity for both OP and ANP in right and left ventricles that was not associated with leukocyte infiltration. In situ hybridization identified cardiomyocytes as the major source of OP mRNA transcripts in these hearts. In contrast, OP immunoreactivity was not detectable in four of five endomyocardial biopsies with normal histology. CONCLUSIONS The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

Angiotensin II causes mesangial cell hypertrophy.

Angiotensin II, a potent vasoconstrictor and known growth factor for vascular smooth muscle cells, has been implicated in the development of glomerulosclerosis. Because mesangial cell growth plays a critical role in the glomerulosclerotic process, the objective of this study was to determine the direct effect of long-term (48-hour) angiotensin II treatment on the growth of cultured murine mesangial cells. Subconfluent, quiescent adult murine mesangial cells were treated for 48 hours with media containing angiotensin II with and without its specific inhibitor losartan. In comparison to cells treated with serum-free medium, cells treated with serum plus insulin demonstrated a significant increase in cell number (1.93 +/- 0.1 times control, p < 0.05), [3H]thymidine incorporation per 10(5) cells (2.29 +/- 0.12 times control, p < 0.05), [3H]leucine incorporation per 10(5) cells (1.81 +/- 0.18 times control, p < 0.05), and total protein content per 10(5) cells (1.65 +/- 0.07 times control, p < 0.05). In contrast, cells treated with angiotensin II (10(-6) M) had no significant increase in cell number (0.84 +/- 0.01 times control) or [3H]thymidine incorporation per 10(5) cells (1.23 +/- 0.12 times control) but demonstrated a significant increase in [3H]leucine incorporation per 10(5) cells (1.61 +/- 0.09 times control) and total protein content per 10(5) cells (1.38 +/- 0.04 times control). Pretreatment with losartan blocked 56% of the angiotensin II-induced increase in [3H]leucine incorporation and 84% of the angiotensin II-induced increase in total protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

Human decidua is a major source of renin.

Plasma prorenin levels are elevated in normal pregnant women. Current evidence suggests renin production by tissues of the uteroplacental unit contribute to this elevation. The purpose of this investigation was to define the source of renin biosynthesis within the human uteroplacental unit and to characterize the renin produced. RNA extraction and Northern blot analysis consistently demonstrated renin mRNA expression in uterine lining both in the pregnant (decidua) and nonpregnant states (endometrium) and in fetal chorion laeve, which is inseparable from the decidua. In contrast, renin mRNA expression was not detected in basal plate and intertwin chorion (which is separate from decidua), amnion, myometrium, or placental villi. The total renin content in decidual homogenates was two- to threefold greater than in endometrial homogenates, and cultured human decidual cells produced significantly more total renin than cultured human endometrial cells, suggesting that pregnancy enhanced renin production by the cells lining the uterus. Immunoblot analysis and [3H]leucine incorporation identified 47,000-mol wt prorenin as the major form of renin produced by cultured human decidual cells. These studies indicate that maternal decidua is the major source of prorenin in the uteroplacental unit.

Integrins, Adhesion, and Cardiac Remodeling

Integrins are heterodimeric cell surface receptors that mediate a cells ability to perceive its environment, respond to changed in its environment, and alter its environment. When activated, these receptors form focal adhesions, which are areas of close attachment of the cells to extracellular matrix proteins in which colocalization of cytoskeletal proteins, intracellular signaling molecules, and growth factor receptors occurs. In cardiac fibroblasts, integrins mediate cell growth and adhesion. Growth factors such as angiotensin II regulate DNA synthesis, protooncogene expression, extracellular matrix production, adhesion, and other actions of cardiac fibroblasts, many of which require integrin activation. In addition to controlling growth factor and hemodynamic effects, regulation ofintegrin activity may be useful to affect cardiac fibrosis and the remodeling process.

Extrarenal renin-secreting tumors : insights into hypertension and ovarian renin production

Although renin-secreting tumors are rare, they must be considered in the differential diagnosis of hypertension associated with hypokalemia, which occurs commonly in the hypertensive population. The finding of an ovarian renin-secreting tumor emphasizes the potential importance of the ovary as an extrarenal source of renin; the local ovarian renin-angiotensin system may play a key role in reproductive function by regulating vascular reactivity, local blood flow, steroidogenesis and other physiologic effects. In the illustrative case presented, a renin-secreting ovarian leiomyosarcoma was obtained from a women who presented with hypertension and hypokalemia. Plasma prorenin levels were markedly elevated. Tumor excision was quickly followed by a fall in prorenin levels and tumor recurrence was accompanied by an increase in prorenin levels. Active renin concentration in the tumor homogenates was similar to that found in kidney homogenates while the tissue prorenin concentration was approximately 20 times that found in kidney tissue. When cultured for up to 4 weeks, ovarian tumor cells secreted greater than 95% prorenin. Immunoblot analysis demonstrated that tumor renin had a molecular weight of 47,000, similar to that of human recombinant prorenin. Immunohistochemical staining of tumor tissue with antibodies against human renal renin at the electron microscopic level demonstrated the presence of renin primarily in membrane-bound vesicles and rarely in dense-core secretory granules. These findings suggest that prorenin in this ovarian tumor was secreted by the constitutive pathway, which is mediated by these amorphous vesicles.

Ovarian renin production in vitro and in vivo: characterization and clinical correlation.

The purpose of this study was to examine the in vitro production of prorenin and active renin by human theca cells and to examine the clinical significance of this production by correlating prorenin and active renin levels with oocyte maturity in follicular fluid samples. Human theca cell cultures were established and were found to produce both prorenin as well as active renin. Androstenedione levels (126 +/- 28 pg/500,000 cells/24-hr incubation) correlated with prorenin levels (8.5 +/- 1.1 ng angiotensin I per milliliter per hour (AI/ml/hr) in culture supernatant (r = 0.61, P less than 0.05). Active renin levels in follicular fluid were higher in stimulated versus spontaneous cycles (359 +/- 67 versus 126 +/- 37 ng AI/ml/hr, P less than 0.05). Renin substrate levels were similar in follicular fluid and in the peripheral serum (1,610 +/- 216 versus 2,160 +/- 490 ng/ml) in spontaneous cycles. Follicular fluid prorenin and active renin did not correlate with oocyte maturity or with steroid levels. The authors conclude that ovarian theca cells produce renin in vitro. However, renin production does not correlate with oocyte maturity or follicular fluid steroids in vivo.

Gradients of prorenin and active renin in ovarian venous and peripheral venous blood samples obtained simultaneously.

Previous studies have demonstrated the presence of prorenin, active renin, and angiotensin II in human follicular fluid. The purpose of this study was to analyze prorenin, active renin, and ovarian steroids in ovarian venous blood and peripheral venous blood samples obtained simultaneously. We studied 10 premenopausal patients undergoing oophorectomy in various phases of the menstrual cycle. Prorenin levels in the ovarian venous effluent were more than twofold higher than levels in peripheral blood, 136.8 +/- 34.1 versus 35.6 +/- 8.3 ng angiotensin 1 per milliliter per hour (p less than 0.01). Active renin levels were also higher in ovarian venous blood than in peripheral venous blood, 12.9 +/- 2.5 versus 8.9 +/- 2.7 ng angiotensin 1 per milliliter per hour, but this difference did not achieve statistical significance (p = 0.07). Prorenin levels correlated with those of active renin in ovarian venous blood (r = 0.76, p less than 0.05), suggesting that prorenin is locally activated. In the peripheral circulation, estradiol levels correlated negatively with prorenin levels (r = -0.73, p less than 0.05), although prorenin levels did not correlate with steroid levels in ovarian venous blood. We conclude that prorenin is produced by the ovary throughout the menstrual cycle and may be locally activated.

Secretion of prorenin by a virilizing ovarian tumor

A 53-year-old normotensive, normokalemic female presented with a 6-month history of virilization. Estradiol, LH, FSH, urinary-free cortisol, and DHEA-S levels were normal. Pelvic ultrasound and computerized tomography were also within normal limits. Her serum testosterone (551 ng/dl; nl, 20-70) and plasma prorenin (124 ng AI/ml/hr; nl, less than 50) levels were elevated. At surgery, a lipoid/steroid cell tumor of the right ovary was removed. Postoperative testosterone and prorenin levels were normal. Ovarian tumor cells, in culture, produced large amounts of prorenin. Immunohistochemistry localized prorenin and/or renin to tumor cells. Determining plasma prorenin levels may be a useful adjunct in diagnosing or following patients with nonepithelial ovarian tumors. A larger clinical study of prorenin levels in patients with such tumors is needed.