Extracellular matrix remodeling in canine and mouse myocardial infarcts.

Extracellular matrix proteins not only provide structural support, but also modulate cellular behavior by activating signaling pathways. Healing of myocardial infarcts is associated with dynamic changes in the composition of the extracellular matrix; these changes may play an important role in regulating cellular phenotype and gene expression. We examined the time course of extracellular matrix deposition in a canine and mouse model of reperfused infarction. In both models, myocardial infarction resulted in fragmentation and destruction of the cardiac extracellular matrix, extravasation of plasma proteins, such as fibrinogen and fibronectin, and formation of a fibrin-based provisional matrix providing the scaffold for the infiltration of granulation tissue cells. Lysis of the plasma-derived provisional matrix was followed by the formation of a cell-derived network of provisional matrix composed of cellular fibronectin, laminin, and hyaluronic acid and containing matricellular proteins, such as osteopontin and osteonectin/SPARC. Finally, collagen was deposited in the infarct, and the wound matured into a collagen-based scar with low cellular content. Although the canine and mouse infarcts exhibited a similar pattern of extracellular matrix deposition, deposition of the provisional matrix was more transient in the mouse infarct and was followed by earlier formation of a mature collagen-based scar after 7-14 days of reperfusion; at the same timepoint, the canine infarct was highly cellular and evolving. In addition, mature mouse infarcts showed limited collagen deposition and significant tissue loss leading to the formation of a thin scar. In contrast, dogs exhibited extensive collagen accumulation in the infarcted area. These species-specific differences in infarct wound healing should be taken into account when interpreting experimental infarction studies and when attempting to extrapolate the findings to the human pathological process.

A murine model of ischemic cardiomyopathy induced by repetitive ischemia and reperfusion.

BACKGROUND: Repetitive brief myocardial ischemia has been implicated in the pathogenesis of the ventricular dysfunction associated with ischemic cardiomyopathy and myocardial hibernation. In this study we examine the effects of repetitive ischemia and reperfusion (I/R) on murine myocardium. METHODS: C57/BL6 mice underwent daily 15 min left anterior descending coronary occlusions followed by reperfusion. After 3, 5, 7, 14, 21 and 28 days, echocardiographic studies were performed, and hearts of I/R and sham-operated animals were processed for histological examination. RESULTS: Histological studies showed no evidence of myocardial necrosis in the ischemic region. Quantitative assessment of collagen revealed a marked persistent interstitial deposition of collagen after seven days I/R in the anterior left ventricular wall (sham 4.6 +/- 2.0 %, I/R 21.5 +/- 6.5 %, p < 0.05). Echocardiographic studies showed persistent regional anterior wall dysfunction in I/R animals. Histological evaluation showed absence of neovessel formation. After discontinuation of the I/R protocol, fibrosis and regional ventricular dysfunction decreased within 60 days. CONCLUSIONS: Repetitive brief murine myocardial I/R induces reversible fibrotic remodeling and ventricular dysfunction, without myocardial infarction and necrosis, and may play a role in the pathogenesis of ischemic cardiomyopathy and myocardial hibernation.

Effect of cellular elements on pressure-velocity relationship in mice.

The effect of cellular elements in the blood on peripheral vascular function in mice was evaluated using the pressure-velocity relationships in the iliac arteries of 5 wild type (WT) and 3 polycythemic (MH) mice. Pressure was obtained using a fluid filled catheter in the left iliac artery and blood velocity was measured in the right iliac artery using a 20 MHz pulsed Doppler probe. The proximal aorta was then occluded for one minute to allow flow velocity to decay to zero. The pressure-velocity relationship in the diastolic phase was determined before and after aortic occlusion. In both groups the pressure-velocity relationship was almost linear and the slopes were similar. However, the extrapolated zero-velocity intercept was significantly higher for the MH than WT mice before (55.4 +/- 4.0 vs. 36.2 +/- 4.1 mmHg, p<0.01) and after occlusion (50.7 +/- 5.5 vs. 23.8 +/- 3.1 mmHg, p<0.01). Hematocrits were 41%+/-3 in WT and 59%+/-3 in MH mice. These data show that cellular elements in the blood alter the pressure-velocity relationships in peripheral vessels of mice.

Brief murine myocardial I/R induces chemokines in a TNF-alpha-independent manner: role of oxygen radicals.

Early chemokine induction in the area at risk of an ischemic-reperfused (I/R) myocardium is first seen in the venular endothelium. Reperfusion is associated with several induction mechanisms including increased extracellular tumor necrosis factor (TNF)-alpha, reactive oxygen intermediate (ROI) species formation, and adhesion of leukocytes to the venular endothelium. To test the hypothesis that chemokine induction in cardiac venules can occur by ROIs in a TNF-alpha-independent manner, and in the absence of leukocyte accumulation, we utilized wild-type (WT) and TNF-alpha double-receptor knockout mice (DKO) in a closed-chest mouse model of myocardial ischemia (15 min) and reperfusion (3 h), in which there is no infarction. We demonstrate that a single brief period of I/R induces significant upregulation of the chemokines macrophage inflammatory protein (MIP) -1 alpha, -1 beta, and -2 at both the mRNA and protein levels. This induction was independent of TNF-alpha, whereas levels of these chemokines were increased in both WT and DKO mice. Chemokine induction was seen predominantly in the endothelium of small veins and was accompanied by nuclear translocation of nuclear factor-kappa B and c-Jun (AP-1) in venular endothelium. Intravenous infusion of the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) initiated 15 min before ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun had no effect. The results suggest that ROI generation in the reperfused myocardium rapidly induces C-C and C-X-C chemokines in the venular endothelium in the absence of infarction or irreversible cellular injury.

Reactive oxygen intermediates induce monocyte chemotactic protein-1 in vascular endothelium after brief ischemia.

Chemokine expression is associated with reperfusion of infarcted myocardium in the setting of tissue necrosis, intense inflammation, and inflammatory cytokine release. The specific synthesis of monocyte chemotactic protein (MCP)-1 mRNA by cardiac venules in reperfused infarcts corresponded to the region where leukocytes normally localize. MCP-1 could be induced by exogenous tumor necrosis factor (TNF)-alpha or by postischemic cardiac lymph containing TNF-alpha. However, the release of TNF-alpha during early reperfusion did not explain the venular localization of MCP-1 induction. To better understand the factors mediating MCP-1 induction, we examined the role of ischemia/reperfusion in a model of brief coronary occlusion in which no necrosis or inflammatory response is seen. Adult mongrel dogs were subjected to 15 minutes of coronary occlusion and 5 hours of reperfusion. Ribonuclease protection assay revealed up-regulation of MCP-1 mRNA only in ischemic segments of reperfused canine myocardium. Pretreatment with the reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine completely inhibited MCP-1 induction. In situ hybridization localized MCP-1 message to small venular endothelium in ischemic areas without myocyte necrosis. Gel shift analysis of nuclear extracts from the ischemic area showed enhanced DNA binding of the transcription factors AP-1 and nuclear factor (NF)-kappaB, crucial for MCP-1 expression, in ischemic myocardial regions. Immunohistochemical staining demonstrated reperfusion-dependent nuclear translocation of c-Jun and NF-kappaB (p65) in small venular endothelium, only in the ischemic regions of the myocardium, that was inhibited by N-(2-mercaptopropionyl)-glycine. In vitro, treatment of cultured canine jugular vein endothelial cells with the reactive oxygen intermediate H2O2 induced a concentration-dependent increase in MCP-1 mRNA levels, which was inhibited by the antioxidant N-acetyl-L-cysteine, a precursor of glutathione, but not pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB and activator of AP-1. In contrast to our studies with infarction, incubation of canine jugular vein endothelial cells with postischemic cardiac lymph did not induce MCP-1 mRNA expression suggesting the absence of cytokine-mediated MCP-1 induction after a sublethal ischemic period. These results suggest that reactive oxygen intermediate generation, after a brief ischemic episode, is capable of inducing MCP-1 expression in venular endothelium through AP-1 and NF-kappaB. Short periods of ischemia/reperfusion, insufficient to produce a myocardial infarction, induce MCP-1 expression, potentially mediating angiogenesis in the ischemic noninfarcted heart.

Cardiac hypertrophy after transplantation is associated with persistent expression of tumor necrosis factor-alpha.

BACKGROUND: The mechanisms that contribute to cardiac allograft hypertrophy are not known; however, the rapid progression and severity of hypertrophy suggest that nonhemodynamic factors may play a contributory role. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine produced in cardiac allografts and capable of producing hypertrophy and fibrosis; therefore, we suggest that TNF-alpha may play a contributory role. Accordingly, the aims of our study were to define the role of systemic hypertension in the development of hypertrophy, characterize the histological determinants of hypertrophy, and characterize the expression of myocardial TNF-alpha after heart transplantation. METHODS AND RESULTS: To separate the effect of hypertension from immune injury in the development of cardiac allograft hypertrophy, we measured the gain in left ventricular mass by 2D echocardiography in heart transplant recipients and lung transplant recipients who developed similar rates of systemic hypertension. The gain in left ventricular mass was 73% in heart transplant recipients and 7% in lung transplant recipients (P<0.0001). By comparing myocardial samples obtained during the first week after transplant and at 1 year, we found that there was a significant increase in total collagen content (P<0.0001), collagen I (P<0.0001), collagen III (P<0.0001), and myocyte size (P<0.0001). These changes were associated with persistent myocardial TNF-alpha expression. CONCLUSIONS: We suggest that the contribution of hypertension to cardiac allograft hypertrophy is minimal and that persistent intracardiac expression of TNF-alpha may contribute to the development of cardiac allograft hypertrophy.

Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival.

Cardiac muscle regeneration after injury is limited by "irreversible" cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis, in vitro and in vivo. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.

Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy is a genetic disease characterized by cardiac hypertrophy, myocyte disarray, interstitial fibrosis, and left ventricular (LV) dysfunction. We have proposed that hypertrophy and fibrosis, the major determinants of mortality and morbidity, are potentially reversible. We tested this hypothesis in beta-myosin heavy chain-Q(403) transgenic rabbits. METHODS AND RESULTS: We randomized 24 beta-myosin heavy chain-Q(403) rabbits to treatment with either a placebo or simvastatin (5 mg. kg(-1). d(-1)) for 12 weeks and included 12 nontransgenic controls. We performed 2D and Doppler echocardiography and tissue Doppler imaging before and after treatment. Demographic data were similar among the groups. Baseline mean LV mass and interventricular septal thickness in nontransgenic, placebo, and simvastatin groups were 3.9+/-0.7, 6.2+/-2.0, and 7.5+/-2.1 g (P<0.001) and 2.2+/-0.2, 3.1+/-0.5, and 3.3+/-0.5 mm (P=0.002), respectively. Simvastatin reduced LV mass by 37%, interventricular septal thickness by 21%, and posterior wall thickness by 13%. Doppler indices of LV filling pressure were improved. Collagen volume fraction was reduced by 44% (P<0.001). Disarray was unchanged. Levels of activated extracellular signal-regulated kinase (ERK) 1/2 were increased in the placebo group and were less than normal in the simvastatin group. Levels of activated and total p38, Jun N-terminal kinase, p70S6 kinase, Ras, Rac, and RhoA and the membrane association of Ras, RhoA, and Rac1 were unchanged. CONCLUSIONS: Simvastatin induced the regression of hypertrophy and fibrosis, improved cardiac function, and reduced ERK1/2 activity in the beta-myosin heavy chain-Q(403) rabbits. These findings highlight the need for clinical trials to determine the effects of simvastatin on cardiac hypertrophy, fibrosis, and dysfunction in humans with hypertrophic cardiomyopathy and heart failure.

Stem cell plasticity in muscle and bone marrow.

Recent discoveries have demonstrated the extraordinary plasticity of tissue-derived stem cells, raising fundamental questions about cell lineage relationships and suggesting the potential for novel cell-based therapies. We have examined this phenomenon in a potential reciprocal relationship between stem cells derived from the skeletal muscle and from the bone marrow. We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5 day in vitro culture were harvested and introduced into each of six lethally irradiated recipients together with distinguishable whole bone marrow cells. Six and twelve weeks later, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56%, indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. Although the identity of the muscle-derived hematopoietic stem cells is still unknown, they may be identical to muscle satellite cells, some of which lack myogenic regulators and could respond to hematopoietic signals. We have also found that stem cells in the bone marrow can contribute to cardiac muscle repair and neovascularization after ischemic injury. We transplanted highly purified bone marrow stem cells into lethally irradiated mice that subsequently were rendered ischemic by coronary artery occlusion and reperfusion. The engrafted stem cells or their progeny differentiated into cardiomyocytes and endothelial cells and contributed to the formation of functional tissue.

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34(-)/low, c-Kit(+), Sca-1(+)) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and alpha-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.

Decreased expression of tumor necrosis factor-alpha and regression of hypertrophy after nonsurgical septal reduction therapy for patients with hypertrophic obstructive cardiomyopathy.

BACKGROUND: Nonsurgical septal reduction therapy (NSRT) is a novel therapeutic strategy for patients with hypertrophic obstructive cardiomyopathy (HOCM). Although the clinical benefits of this technique appear to be clear, the structural and functional changes that lead to improvements in cardiac function are not completely defined. In these studies, we sought to define the effect of NSRT on myocardial function as well as various markers of hypertrophy including the expression of tumor necrosis factor (TNF)-alpha, a cytokine capable of producing fibrosis, left ventricular hypertrophy (LVH), and cardiomyopathy. METHODS AND RESULTS: We performed endomyocardial biopsies of the RV side of the septum and echocardiograms on 15 HOCM patients at baseline and after successful NSRT. Comparative analysis on paired myocardial samples were performed to determine the effects of NSRT on LVH, end-diastolic volume and chamber stiffness, myocyte size, collagen content, and TNF-alpha levels. At baseline, myocardial TNF-alpha levels were increased in all patients. After NSRT, myocyte size, collagen content, and TNF-alpha were significantly decreased. These changes were accompanied by an increase in left ventricular volumes and a reduction in LVH and chamber stiffness. CONCLUSIONS: We suggest that pressure overload in HOCM patients contributes to the development of hypertrophy. These data provide the initial experimental evidence to suggest that TNF-alpha may play a pathogenetic role in the hypertrophy of pressure overload.

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice.

P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.

Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb).

OBJECTIVE: The purpose of this study is to examine the cellular content of healing myocardial infarcts and study the phenotypic characteristics of fibroblasts during scar formation utilizing a canine model of coronary occlusion and reperfusion. METHODS: Ischemia/Reperfusion experiments were performed in dogs undergoing 1 h of coronary occlusion followed by reperfusion intervals ranging from 5 h to 28 days. Fibrotic and control areas were studied using immunohistochemistry. RESULTS: The healing ischemic and reperfused myocardium demonstrated significant proliferative activity peaking after 3 to 7 days of reperfusion, predominantly in myofibroblasts. The numbers of proliferating cells decreased during the maturation phase of the scar (PCNA index: 13.7+/-2.25% at 5 days vs. 4.8+/-1.1% at 28 days; P<0.05, n=5). During the proliferative phase of healing (3-7 days) alpha-smooth muscle actin (alpha-SMAc) expression was markedly increased in the fibrotic areas. alpha-SMAc predominantly localized in myofibroblasts which were vimentin positive, smooth muscle myosin, calponin and desmin negative. We examined expression of smooth muscle myosin heavy chain isoforms in myofibroblasts infiltrating the healing areas and found a marked induction of the embryonal isoform of myosin heavy chain (SMemb) in alpha-SMAc positive spindle shaped cells in the border of the scar. Myofibroblasts did not express SM2, a marker for mature smooth muscle cells. In contrast myocardial arterioles were positive for SM2, but did not express SMemb. CONCLUSIONS: Healing myocardial infarcts undergo rapid changes in their content of myofibroblasts. During the proliferative phase fibroblasts undergo phenotypic changes leading to expression of contractile proteins such as alpha-SMAc, and production of SMemb, a marker for dedifferentiated smooth muscle cells. Expression of embryonic isoforms indicates dedifferentiation and allows the myofibroblast pool to serve as a versatile cell population, assuming different phenotypes depending on the physiological needs.

Hemodynamic changes in apolipoprotein E-knockout mice.

Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P < 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P < 0.001), and pulse-wave velocity (+13%, P < 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios.

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury.

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.

Interleukin 6 induction in the canine myocardium after cardiopulmonary bypass.

OBJECTIVE: Interleukin 6 is a proinflammatory cytokine with a plasma concentration that has been noted to increase in response to cardiopulmonary bypass. The source of interleukin 6 after cardiopulmonary bypass is unknown. This study examined the myocardium as a potential source of interleukin 6 in this context. METHODS: Dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After rewarming, they were reperfused with the chest open for either 3 (n = 4) or 6 (n = 4) hours, at the end of which myocardial samples were obtained. Four additional animals undergoing open thoracotomy without bypass served as time-matched controls. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization were used to examine the myocardium for the induction of interleukin 6 and intercellular adhesion molecule-1. RESULTS: Northern blot analysis and reverse transcriptase-polymerase chain reaction demonstrated a marked increase in myocardial interleukin 6 messenger RNA in 3 of 4 dogs at 3 hours after bypass and 3 of 4 dogs at 6 hours after bypass, which was not present in sham-bypass control animals. Northern blots at 3 hours after cardiopulmonary bypass also demonstrated myocardial intercellular adhesion molecule-1 induction. In situ hybridization studies confirmed that cardiac myocytes were a principal source of interleukin 6 messenger RNA early after cardiopulmonary bypass. Northern blots of messenger RNA extracted from isolated neutrophils and mononuclear leukocytes obtained from blood samples before bypass, at the end of bypass, and 3 hours after bypass failed to demonstrate interleukin 6 induction. CONCLUSION: Despite protection with cold cardioplegic arrest, the myocardium was a significant source of interleukin 6 synthesis after cardiopulmonary bypass. Local production of interleukin 6 may play a pivotal role in postoperative myocardial function.

Osmotically relevant membrane signaling complex: association between HB-EGF, beta(1)-integrin, and CD9 in mTAL.

The integral membrane proteins cluster of differentiation-9 (CD9), beta(1)-integrin, and heparin-binding epidermal growth factor-like (HB-EGF) exist in association in many cell lines and are linked to intracellular signaling mechanisms. Two of the proteins (CD9 and beta(1)-integrin) are induced by hypertonicity, suggesting that their related signaling processes may be relevant to osmotic stress. The validity of this hypothesis rests upon coexpression and physical association between these molecules in nephron segments that are normally exposed to high and variable ambient osmolality. In this work, we show that CD9 and beta(1)-integrin are induced in rat kidney medulla after dehydration. Immunohistochemistry and immunoprecipitation studies show that CD9, HB-EGF, and beta(1)-integrin are coexpressed and physically associated in medullary thick ascending limbs (mTAL), nephron segments that are normally exposed to high and variable extracellular osmolality. Our findings are consistent with the existence of a cluster of integral membrane proteins in mTAL that may initiate or modulate osmotically relevant signaling pathways.

A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies.

Reperfusion of the ischemic myocardium is associated with a cytokine cascade that reflects a cellular response to injury. We studied this cascade in the mouse and found that acute surgical trauma in sham-operated animals obscured early changes in cytokine induction that occur during myocardial ischemia-reperfusion (MI/R). Therefore, we utilized a new implantable device that allows occlusion and reperfusion of the left anterior descending coronary artery in a closed-chest mouse at any time after instrumentation. Induction of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha mRNA in the whole heart was examined by RNase protection assay and quantitated by Phosphor- Imager. At 3 h after instrumentation, levels of IL-6 mRNA in sham-operated animals increased above those of control naive hearts, whereas this increase did not occur until after 1 day for TNF-alpha mRNA. The surgical trauma led to exaggeration of I/R cytokine induction with greater variance in response. At 3 days and 1 wk after instrumentation, levels of both IL-6 and TNF-alpha mRNA in sham-operated animals were comparable to those of naive hearts and induction responses in I/R were much less variant. We also found that 1 h of ischemia and 2 h of reperfusion at all time points of recovery (i.e., 3 h and 1, 3, and 7 days after instrumentation) led to a significant increase in IL-6 and TNF-alpha mRNA levels. In addition, 3 h of permanent occlusion, which did not induce any mRNA increase after 1 wk postinstrumentation, caused marked upregulation of IL-6 mRNA in an acutely prepared animal. This study of early cytokine responses evoked by MI/R highlights the need for dissipation of acute surgical trauma by using a chronic, closed-chest mouse preparation.