Using a high-frequency linear transducer (15L8), we studied 1) the feasibility of performing echocardiography in nonanesthetized mice compared with mice given pentobarbital sodium (Pento) or a mixture of ketamine and xylazine and 2) the feasibility of echocardiographic evaluation of left ventricular (LV) hypertrophy, dilatation, and function in mice with two-kidney, one-clip hypertension or myocardial infarction (MI). Heart rate (HR) in awake mice was 658 +/- 9 beats/min; Pento and ketamine plus xylazine reduced HR to 377 +/- 11 and 293 +/- 19 beats/min, respectively, associated with a significant decrease in shortening fraction (SF), ejection fraction (EF), and cardiac output (CO) and an increase in LV end-diastolic (LVEDD) and end-systolic dimensions (LVESD). Mice with 4 wk of two-kidney, one-clip hypertension had increased LV mass (15.62 +/- 0. 62 vs. 22.17 +/- 1.79 mg) without altered LV dimensions, SF, EF, or CO. Mice studied 4 wk post-MI exhibited obvious LV dilatation and systolic dysfunction, as evidenced by increased LVEDD and LVESD and decreased SF, EF, and CO. Our findings clearly show the adverse impact of anesthesia on basal cardiac function and the difficulty in interpreting data obtained from anesthetized mice. We believe this is the first study to demonstrate the feasibility of using echocardiography to assess cardiovascular function in the nonanesthetized mouse.

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-Inflammation may play an important role in the pathogenesis of cardiac fibrosis in heart failure (HF) after myocardial infarction (MI). N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring antifibrotic peptide whose plasma concentration is increased 4-to 5-fold by angiotensin-converting enzyme inhibitors. We tested the hypothesis that in rats with HF after MI, Ac-SDKP acts as an anti-inflammatory cytokine, preventing and also reversing cardiac fibrosis in the noninfarcted area (reactive fibrosis), and thus affording functional improvement. We found that Ac-SDKP significantly decreased total collagen content in the prevention group from 23.7Ϯ0.9 to 15.0Ϯ0.7 g/mg and in the reversal group from 22.6Ϯ2. , PϽ0.01 (reversal). Ac-SDKP did not alter either blood pressure or left ventricular hypertrophy (LVH); however, it depressed systolic cardiac function in the prevention study while having no significant effect in the reversal group. We concluded that Ac-SDKP has an anti-inflammatory effect in HF that may contribute to its antifibrotic effect; however, this decrease in fibrosis without changes in LVH was not accompanied by an improvement in cardiac function. Key Words: rat Ⅲ myocardial infarction Ⅲ cardiac function Ⅲ collagen Ⅲ macrophages Ⅲ transforming growth factor-␤ N -acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring antifibrotic peptide whose plasma concentration is increased 4-to 5-fold by angiotensin-converting enzyme inhibitors. Ac-SDKP is released from its precursor thymosin-␤ 4 , which is present in most cells. 1 It inhibits pluripotent hematopoietic stem cell and hepatocyte proliferation by halting entry into the S phase of the cell cycle, maintaining cells in the G 0 /G 1 phase and thereby helping control their proliferation. [2][3][4] We have shown that in vitro Ac-SDKP inhibited cardiac fibroblast proliferation and collagen synthesis, 5 while in vivo it prevented collagen deposition in the left ventricle (LV) and kidneys in rats with aldosterone-salt hypertension and renovascular hypertension. 5,6 This decrease in collagen deposition was associated with a reduced number of proliferating cell nuclear antigen (PCNA)-positive cells, a marker of cell proliferation. These effects of Ac-SDKP occurred without changes in blood pressure or cardiomyocyte hypertrophy. Our studies suggest that one of the mechanisms by which Ac-SDKP prevents fibrosis is by inhibiting fibroblast proliferation and collagen synthesis. It is also known that inflammation plays a central role in the pathogenesis of interstitial and perivascular cardiac fibrosis in heart failure (HF) post-myocardial infarction (MI). Fibrosis is often co-localized with macrophages, which release cytokines such as transforming growth factor-␤ (TGF-␤) that play a crucial role in myocardial fibrosis. 7 There is evidence that Ac-SDKP inhibits TGF-␤ signal transduction through suppression of Smad2 phosphorylation. 8,9 However, it is not known whether it also inhibits the expression of TGF-␤.In the present study,...

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-...

Abstract-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a ubiquitous tetrapeptide hydrolyzed almost exclusively by angiotensin-converting enzyme (ACE). Chronic treatment with Ac-SDKP decreases cardiac and renal fibrosis and inflammatory cell infiltration in hypertensive rats. However, very little is known about endogenous synthesis of Ac-SDKP, except that thymosin-␤ 4 may be the most likely precursor. Two enzymes are potentially able to release Ac-SDKP from thymosin-␤ 4 : prolyl oligopeptidase (POP) and endoproteinase asp-N. POP is widely present and active in several tissues and biological fluids, whereas endoproteinase asp-N appears to be lacking in mammals. Therefore, we hypothesized that POP is the main enzyme involved in synthesizing the antifibrotic peptide Ac-SDKP. We investigated in vitro and in vivo production of Ac-SDKP. Using kidney cortex homogenates, we observed that Ac-SDKP was generated in a time-dependent manner in the presence of exogenous thymosin-␤ 4 , and this generation was significantly inhibited by several POP inhibitors (POPi), Z-prolyl-prolinal, Fmoc-prolyl-pyrrolidine-2-nitrile, and S17092. Long-term administration of S17092 in rats significantly decreased endogenous levels of Ac-SDKP in the plasma (from 1.76Ϯ0.2 to 1.01Ϯ0.1 nM), heart (from 2.31Ϯ0.21 to 0.83Ϯ0.09 pmol/mg protein), and kidneys (from 5.62Ϯ0.34 to 2.86Ϯ0.76 pmol/mg protein). As expected, ACE inhibitors significantly increased endogenous levels of Ac-SDKP in the plasma, heart, and kidney, whereas coadministration of POPi prevented this increase. We concluded that POP is the main enzyme responsible for synthesis of the antifibrotic peptide

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