prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin.

Background-Angiotensin-converting enzyme (ACE) inhibitors are valuable agents for the treatment of hypertension, heart failure, and other cardiovascular and renal diseases. The cardioprotective effects of ACE inhibitors are mediated by blockade of both conversion of angiotensin (Ang) I to Ang II and kinin hydrolysis. Here, we report a novel mechanism that may explain the cardiac antifibrotic effect of ACE inhibition, involving blockade of the hydrolysis of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Methods and Results-To study the role of Ac-SDKP in the therapeutic effects of the ACE inhibitor captopril, we used a model of Ang II-induced hypertension in rats treated with the ACE inhibitor either alone or combined with a blocking monoclonal antibody (mAb) to Ac-SDKP. These hypertensive rats had left ventricular hypertrophy (LVH) as well as increases in cardiac fibrosis, cell proliferation, transforming growth factor-␤ (TGF-␤) expression, and phosphorylation of Smad2 (P-Smad2), a signaling mediator of the effects of TGF-␤. The ACE inhibitor did not decrease either blood pressure or LVH; however, it significantly decreased LV collagen from 13.3Ϯ0.9 to 9.6Ϯ0.6 g/mg dry wt (PϽ0.006), and this effect was blocked by the mAb (12.1Ϯ0.6; PϽ0.034, ACE inhibitor versus ACE inhibitorϩmAb). In addition, analysis of interstitial collagen volume fraction and perivascular collagen (picrosirius red staining) showed a very similar tendency. Likewise, the ACE inhibitor significantly decreased LV monocyte/macrophage infiltration, cell proliferation, and TGF-␤ expression, and these effects were blocked by the mAb. Ang II increased Smad2 phosphorylation 3.2Ϯ0.9-fold; the ACE inhibitor lowered this to 0.6Ϯ0.1-fold (PϽ0.001), and the mAb blocked this decrease to 2.1Ϯ0.3 (PϽ0.001, ACE inhibitor versus ACE inhibitorϩmAb). Similar findings were seen when the ACE inhibitor was replaced by Ac-SDKP. Conclusions-We concluded that in Ang II-induced hypertension, the cardiac antifibrotic effect of ACE inhibitors is a result of the inhibition of Ac-SDKP hydrolysis, resulting in a decrease in cardiac cell proliferation (probably fibroblasts), inflammatory cell infiltration, TGF-␤ expression, Smad2 activation, and collagen deposition.

Abstract-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits not only hematopoietic cell proliferation but also fibroblast proliferation and collagen synthesis in vitro. Ac-SDKP also prevents collagen deposition and cell proliferation in the left ventricle (LV) in rats with renovascular hypertension (renin dependent). However, it is not clear whether Ac-SDKP has similar effects in a model of renin-independent hypertension (aldosterone-salt). Using a hypertensive rat model of cardiac and renal fibrosis created by chronic elevation of circulating aldosterone (ALDO) levels, we examined the effect of Ac-SDKP on blood pressure, cardiac and renal fibrosis and hypertrophy, and proliferating cell nuclear antigen (PCNA) expression in the LV and left kidney. Uninephrectomized rats were divided into 4 groups: (1) controls that received tap water, (2) rats that received ALDO (0.75 g/h SC) and 1% NaCl/0.2% KCl in drinking water (ALDO-salt), (3) rats that received ALDO-salt plus Ac-SDKP 400 g · kg Ϫ1 · day Ϫ1 SC, and (4) rats that received ALDO-salt plus Ac-SDKP 800 g · kg Ϫ1 · d Ϫ1 SC. After 6 weeks of treatment, the ALDO-salt group was found to have significantly increased blood pressure with decreased body weight and plasma renin concentration (PϽ0.05), LV and renal hypertrophy as well as renal injury, significantly increased collagen content in both ventricles and kidney as well as increased collagen volume fraction in the LV (PϽ0.0001), and significantly increased interstitial and perivascular PCNA-positive cells in the LV and kidney (PϽ0.0001). Ac-SDKP at 800 g · kg Ϫ1 · d Ϫ1 markedly prevented cardiac and renal fibrosis (PϽ0.005) without affecting blood pressure or organ hypertrophy. It also suppressed PCNA expression in the LV and kidney in a dose-dependent manner. We concluded that Ac-SDKP prevents increased collagen deposition and cell proliferation in the heart and kidney in ALDO-salt hypertensive rats. Because ACE inhibitors increase plasma and tissue Ac-SDKP and decrease cardiac and renal fibrosis, we speculate that Ac-SDKP may participate in the antifibrotic effect of ACE inhibitors. Key Words: aldosterone Ⅲ hypertension, mineralocorticoid Ⅲ collagen Ⅲ heart Ⅲ kidney N -acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation 1,2 that is normally present in human plasma and circulating mononuclear cells 3 and is cleaved to an inactive form by the NH 2 -terminal catalytic domain of ACE. 4 There have been reports that administration of captopril, an ACE inhibitor (ACEi), prevented degradation of endogenous Ac-SDKP and raised its circulating and urine concentrations. 4 -6 However, the antiproliferative effects of Ac-SDKP are not limited to hematopoietic cells. We found that Ac-SDKP not only inhibited adult rat cardiac fibroblast proliferation and collagen synthesis in vitro but also reduced the number of proliferating cell nuclear antigen (PCNA)-positive cells and prevented enhanced collagen deposition in the left ventricle (LV) in 2-kidney, 1-...

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell entry into the S phase of the cell cycle and is normally present in human plasma. Ac-SDKP is exclusively hydrolyzed by ACE, and its plasma concentration is increased 5-fold after ACE inhibition in humans. We examined the effect of 0.05 to 100 nmol/L Ac-SDKP on 24-hour 3 H-thymidine incorporation (DNA synthesis) by cardiac fibroblasts both in the absence and presence of 5% FCS. Captopril (1 mol/L) was added in all cases to prevent the degradation of Ac-SDKP. Treatment of cardiac fibroblasts with 5% FCS increased thymidine incorporation from a control value of 12 469Ϯ594 to 24 598Ϯ1051 cpm (PϽ0.001). Cotreatment with 1 nmol/L Ac-SDKP reduced stimulation to control levels (10 373Ϯ200 cpm, PϽ0.001). We measured hydroxyproline content and incorporation of 3 H-proline into collagenous fibroblast proteins and found that Ac-SDKP blocked endothelin-1 (10 Ϫ8 mol/L)-induced collagen synthesis in a biphasic and dose-dependent manner, causing inhibition at low doses, whereas high doses had little or no effect. It also blunted the activity of p44/p42 mitogen-activated protein kinase in a biphasic and dose-dependent manner in serum-stimulated fibroblasts, suggesting that the inhibitory effect of DNA and collagen synthesis may depend in part on blocking mitogen-activated protein kinase activity. Participation of p44/p42 in collagen synthesis was confirmed, because a specific inhibitor for p44/p42 activation (PD 98059, 25 mol/L) was able to block endothelin-1-induced collagen synthesis, similar to the effect of Ac-SDKP. The fact that Ac-SDKP inhibits DNA and collagen synthesis in cardiac fibroblasts suggests that it may be an important endogenous regulator of fibroblast proliferation and collagen synthesis in the heart. Ac-SDKP may participate in the cardioprotective effect of ACE inhibitors by limiting fibroblast proliferation (and hence collagen production), and therefore it would reduce fibrosis in patients with hypertension.

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBPinduced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-␣ by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-␣ release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 Ϯ 1.06 mean fluorescent intensity for vehicle and 10.63 Ϯ 0.35 for Ac-SDKP; P Ͻ 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colonystimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-␣ by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension. macrophages; inflammation; activation; angiotensin II HYPERTENSION LEADS TO CARDIAC, renal, and vascular damage. The mechanisms of target organ damage have not been fully elucidated. There is evidence that inflammation contributes to end organ damage (20). We have shown that in ANG IIinduced hypertension (32), as well as in renovascular hypertension (25, 35), mineralocorticoid-salt hypertension (26, 28), spontaneously hypertensive rats (9), and heart failure postmyocardial infarction (24), N-acetyl-seryl-aspartyl-lysyl-proline

Background-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation. Ac-SDKP plasma concentration is increased 5-fold after angiotensin-converting enzyme inhibition. Here we studied the effect of Ac-SDKP on monocyte/macrophage infiltration, fibroblast proliferation, and collagen deposition in the rat heart in renovascular hypertension. Methods and Results-We investigated whether long-term Ac-SDKP administration would prevent left ventricular (LV) hypertrophy and interstitial collagen deposition in rats with 2-kidney, 1-clip (2K-1C) hypertension. Ac-SDKP (400 g · kg Ϫ1 · d Ϫ1 ) did not affect development of hypertension. Mean blood pressure was similar in rats with 2K-1C hypertension whether they were given vehicle or Ac-SDKP and was higher than in controls. Both LV weight and cardiomyocyte size were significantly increased in rats with 2K-1C hypertension compared with controls and were unaffected by Ac-SDKP. Proliferating cell nuclear antigen-and monocyte/macrophage-positive cells were increased in the LV of 2K-1C hypertensive rats; this increase was significantly blunted by Ac-SDKP (PϽ0.001). LV interstitial collagen fraction was also increased in 2K-1C hypertensive rats given vehicle (10.1Ϯ0.8%) compared with sham (5.3Ϯ0.1%, PϽ0.0001), and this increase was prevented by Ac-SDKP (5.4Ϯ0.4%, PϽ0.001). Conclusions-Ac-SDKP inhibited monocyte/macrophage infiltration, cell proliferation, and collagen deposition in the LV of hypertensive rats without affecting blood pressure or cardiac hypertrophy, suggesting that it may be partly responsible for the cardioprotective effect of angiotensin-converting enzyme inhibitors.

We concluded that Ac-SDKP may be an important mediator of the anti-inflammatory and antifibrotic effects of ACEi in hypertension independent of its hemodynamic effects.

Elevated interleukin-4 (IL-4) levels are associated with cardiac fibrosis in hypertension and heart failure in both patients and experimental animals. We hypothesized that chronically elevated IL-4 induces cardiac fibrosis, resulting in a predisposition of the heart to angiotensin II–induced damage. Wild-type Balb/c (WT, high circulating IL-4) and IL-4–deficient Balb/c mice (IL-4−/−) were used. WT mice exhibited cardiac fibrosis (evidenced by an increase in expression of procollagen genes/interstitial collagen fraction), enlarged left ventricle chamber, and declined cardiac function associated with a greater number of mast cells and macrophages in the heart compared with IL-4−/−. In contrast, IL-4−/− mice had normal cardiac architecture/function while showing a 57.9% reduction in heart interstitial collagen compared with WT, despite elevated proinflammatory cytokines in heart tissue. In response to angiotensin II administration, IL-4−/− had reduced interstitial myocardial fibrosis and were protected from developing dilated cardiomyopathy, which was seen in WT mice. This was associated with increased macrophage infiltration into the hearts of WT mice, despite a similar degree of hypertension and increased cardiac transforming growth factor-β1 in both groups. In vitro data demonstrated that IL-4 upregulates procollagen genes and stimulates collagen production in mouse cardiac fibroblasts. This process is mediated by signal transducer and activator of transcription 6 signaling pathway via IL-4 receptor alpha. This study not only establishes a causal relationship between IL-4 and cardiac fibrosis/dysfunction, but also reveals a critical role for IL-4 in angiotensin II–induced cardiac damage. IL-4 could serve as an additional target for the treatment of cardiac fibrosis.