Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho-associated coiled-coil protein kinase 1.

We have previously found that in failing human hearts, Rho-associated coiled-coil protein kinase 1 (ROCK1) is processed by caspase-3 into an active isoform, ROCKΔ1. The purpose of the current investigation was to elucidate the pathological consequences of truncated ROCK1 accumulation in the heart, the associated molecular mechanism of ROCKΔ1-mediated cardiac phenotype, and the molecular signaling between Rho kinase activation in cardiomyocytes and extracellular matrix response. We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic the situation observed in human heart disease, whereas an additional kinase-deficient mouse was generated as a control. The ROCKΔ1 transgenic mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic hearts displayed activated TGFß1 and NF-κB signaling and a release of a subset of cytokines and were susceptible to angiotensin II stress. Treatment with a Rho kinase inhibitor attenuated the fibrotic phenotype. Cardiac fibroblasts differentiated into myofibroblasts when cocultured with transgenic cardiomyocytes but not with wild-type cardiomyocytes. Inhibitors of Rho kinase as well as TGFßR1 and NF-κB decreased these effects. The serum response factor-dependent TGFß1 regulation was shown to be responsible for the Rho kinase-mediated activation of TGFß1 signaling. We conclude that ROCKΔ1 is a novel fibrotic factor. Activation of TGFß1 and NF-κB signaling contributes to the Rho kinase-mediated pathological fibrosis.

Hypothermia induced by adenosine 5'-monophosphate attenuates early stage injury in an acute gouty arthritis rat model.

To investigate whether the hypothermia induced by Adenosine 5'-Monophosphate (5'-AMP) could attenuate early stage injury in a rat acute gouty arthritis model. Ankle joint injection with monosodium urate monohydrate crystals (MSU crystals) in hypothermia rat model which was induced by 5'-AMP and then observe whether hypothermia induced by 5'-AMP could be effectively inhibit the inflammation on acute gouty arthritis in rats. AMP-induced hypothermia has protective effects on our acute gouty arthritis, which was demonstrated by the following criteria: (1) a significant reduction in the ankle swelling (p < 0.001); (2) a significant decrease in the occurrence of leukocyte infiltration and mild hemorrhage; (3) a significant reduction in the presence of serum Interleukin-1ß (IL-1ß, p < 0.001) and metalloproteinase-9 (MMP-9, p < 0.001); and (4) a significant inhibition in the Nuclear Factor -κappaB (NF-κB) activity (p < 0.001). AMP-induced hypothermia could inhibit acute inflammation reaction and protect the synovial tissue against acute injury in a rat acute gouty arthritis model.

A role of erythrocytes in adenosine monophosphate initiation of hypometabolism in mammals.

Biochemical and mechanistic aspects into how various hypometabolic states are initiated in mammals are poorly understood. Here, we show how a state of hypometabolism is initiated by 5'-AMP uptake by erythrocytes. Wild type, ecto-5'-nucleotidase-deficient, and adenosine receptor-deficient mice undergo 5'-AMP-induced hypometabolism in a similar fashion. Injection of 5'-AMP leads to two distinct declining phases of oxygen consumption (VO(2)). The phase I response displays a rapid and steep decline in VO(2) that is independent of body temperature (T(b)) and ambient temperature (T(a)). It is followed by a phase II decline that is linked to T(b) and moderated by T(a). Altering the dosages of 5'-AMP from 0.25- to 2-fold does not change the phase I response. For mice, a T(a) of 15 degrees C is effective for induction of DH with the appropriate dose of 5'-AMP. Erythrocyte uptake of 5'-AMP leads to utilization of ATP to synthesize ADP. This is accompanied by increased glucose but decreased lactate levels, suggesting that glycolysis has slowed. Reduction in glycolysis is known to stimulate erythrocytes to increase intracellular levels of 2,3-bisphosphoglycerate, a potent allosteric inhibitor of hemoglobin's affinity for oxygen. Our studies showed that both 2,3-bisphosphoglycerate and deoxyhemoglobin levels rose following 5'-AMP administration and is in parallel with the phase I decline in VO(2). In summary, our investigations reveal that 5'-AMP mediated hypometabolism is probably triggered by reduced oxygen transport by erythrocytes initiated by uptake of 5'-AMP.

Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice.

There are conflicting data about gender differences in cardiac function after myocardial infarction (MI), including cardiac rupture and mortality. Using a mouse model of MI, we recently found that the cardiac rupture rate during the first week after MI was significantly lower in females than in males, suggesting that females have attenuated structural remodeling. Thus in this study, we attempted to determine whether: a) females have attenuated remodeling and faster healing during the early phase post-MI, and b) females have better cardiac function and outcome during the chronic phase compared to males. MI was induced in 12-week-old male and female C57BL/6J mice. Signs of early remodeling, including cardiac rupture, infarct expansion, inflammatory response, and collagen deposition, were studied during the first 2 weeks post-MI. Left ventricular remodeling and function were followed for 12 weeks post-MI. We found that males had a higher rate of cardiac rupture, occurring mainly at 3 to 5 days of MI and associated with a higher infarct expansion index. Neutrophil infiltration at the infarct border was more pronounced in males than females during the first days of MI, which were also characterized by increased MMP activity. However, the number of infiltrating macrophages was significantly higher in females at day 4. During the chronic phase post-MI, males had significantly poorer LV function, more prominent dilatation and significant myocyte hypertrophy compared to females. In conclusion, males have delayed myocardial healing, resulting in cardiac rupture, and the survivors have poorer cardiac function and pronounced maladaptive remodeling, whereas females show a better outcome during the development of HF.

Temporal changes in matrix metalloproteinase expression and inflammatory response associated with cardiac rupture after myocardial infarction in mice.

We previously found that male mice with myocardial infarction (MI) had a high rate of cardiac rupture, which generally occurred at 3 to 5 days after MI. Since matrix metalloproteinases (MMPs) play an important role in infarct healing, tissue repair and extracellular matrix (ECM) remodeling post-MI, we studied the temporal relationship of MMP expression and inflammatory response to cardiac rupture after acute MI. Male C57BL/6J mice were subjected to MI (induced by ligating the left anterior descending coronary artery) and killed 1, 2, 4, 7 or 14 days after MI. MMP-2 and MMP-9 activity in the heart were measured by zymography. Collagen content was measured by hydroxyproline assay. We found that after MI, MMP-9 activity increased as early as 1 day and reached a maximum by 2-4 days, associated with a similar increase in neutrophil and macrophage infiltration in the infarct area. MMP-2 started to increase rapidly within 4 days, reaching a maximum by 7 days and remaining high even at 14 days. Intense macrophage infiltration appeared by 4 days after MI and then gradually decreased within 7 to 14 days. Collagen content was unchanged until 4 days after MI, at which point it increased and remained high thereafter. Our data suggest that in mice, overexpression of MMP-2 and MMP-9 (possibly expressed mainly by neutrophils and macrophages) may lead to excessive ECM degradation in the early phase of MI, impairing infarct healing and aggravating early remodeling which in turn causes cardiac rupture.

Metabolite Profiling of 5'-AMP-Induced Hypometabolism.

We have demonstrated that 5'-adenosine-monophosphate (5'-AMP) can be used to induce deep hypometabolism in mice and other non-hibernating mammals. This reversible 5'-AMP induced hypomatabolism (AIHM) allows mice to maintain a body temperature about 1°C above the ambient temperature for several hours before spontaneous reversal to euthermia. Our biochemical and gene expression studies suggested that the molecular processes involved in AIHM behavior most likely occur at the metabolic interconversion level, rather than the gene or protein expression level. To understand the metabolic processes involved in AIHM behavior, we conducted a non-targeted comparative metabolomics investigation at multiple stages of AIHM in the plasma, liver and brain of animals that underwent AIHM. Dozens of metabolites representing many important metabolic pathways were detected and measured using a metabolite profiling platform combining both LC-MS and GC-MS. Our findings indicate that there is a widespread suppression of energy generating metabolic pathways but lipid metabolism appears to be minimally altered. Regulation of carbohydrate metabolites appears to be the major way the animal utilizes energy in AIHM and during the following recovery process. The 5'-AMP administered has largely been catabolized by the time the animals have entered AIHM. During AIHM, the urea cycle appears to be functional, helping to avoid ammonia toxicity. Of all tissues studied, brain's metabolite flux is the least affected by AIHM.

5'- Adenosine monophosphate induced hypothermia reduces early stage myocardial ischemia/reperfusion injury in a mouse model.

Early intervention using hypothermia treatment has been shown to reduce early inflammation, apoptosis and infarct size in animal models of cardiac ischemia/reperfusion. We have shown that 5'-adenosine monophosphate (5'-AMP) can induce a reversible deep hypothermia in mammals. We hypothesize that 5'-AMP-induced hypothermia (AIH) may reduce ischemic/reperfusion damage following myocardial infarct. C57BL/6J male mice were subjected to myocardial ischemia by ligating the left anterior descending coronary artery (LAD) followed by reperfusion. Compared to euthermic controls, mice given AIH treatment exhibited significant inhibition of neutrophil infiltration and a reduction in matrix metallopeptidase 9 (MMP-9) expressions in the infarcted myocardium. A decrease in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in the left ventricle myocardium were also observed. The overall infarct size of the heart was significantly smaller in AIH treated mice. Myocardial ischemia in mice given 5'-AMP without hypothermia had similar ischemia/reperfusion injuries as the euthermic control. Thus, the AIH cardio-protective effects were primarily hypothermia based.

Cysteine residues in CUB-1 domain are critical for ADAMTS13 secretion and stability.

Upon stimulation, endothelial cells release von Willebrand factor (VWF) enriched in ultra-large (UL) forms that are rapidly cleaved by ADAMTS13. The zinc metalloprotease fits in the consensus for members of the ADAMTS family, but also contains two unique C-terminal CUB domains. There are five and two cysteine residues in the CUB-1 and CUB-2 domains, respectively, instead of four as deducted from the consensus. In this study, we investigated the role of cysteine residues in the CUB-1 domain in ADAMTS13 synthesis and activity. CUB-1 and cysteine mutations were expressed in mammalian cell lines and examined for synthesis, secretion, stability, and VWF-cleaving activity. When expressed as an isolated domain, CUB-1, but not CUB-2, covalently aggregated. Converting any of the four cysteines that fit in the CUB consensus (C1192, C1213, C1236 and C1254) reduced the secretion of the mutants to the conditioned medium, but not to extracellular matrix. The mutations also resulted in a moderate increase in proteolytic degradation and decrease in cleaving VWF under static, but not flowing conditions. In contrast, replacing C1275, which was found to be in the thiol form, with a serine residue prevented covalent aggregation of CUB-1, but had no effect on secretion and VWF-cleaving activity. C1275S was also markedly resistant to proteolytic degradation. The data illustrate the importance of consensus cysteines in the secretion and proteolytic activity of ADAMTS13. They also identify an ADAMTS-13 mutant that is resistant to proteolytic degradation, while maintaining a normal VWF-cleaving activity.

Effects of naturally occurring mutations in CUB-1 domain on synthesis, stability, and activity of ADAMTS-13.

INTRODUCTION: Upon stimulation, endothelial cells release von Willebrand factor (VWF) in the unusually large (UL) and hyperactive forms that are rapidly cleaved by ADAMTS-13. Mutations in the ADAMTS13 gene result in ULVWF-mediated thrombosis found in patients with familial thrombotic thrombocytopenia purpura (TTP). ADAMTS-13 fits in the consensus of the ADAMTS family metalloproteases, but also contains two unique C- terminal CUB domains. Studying mutations in CUB domains could provide insights into the functional role of these domains. METHODS: Three naturally occurring mutations (C1213Y, W1245del and K1256FS) in the CUB-1 domain found in patients with TTP were expressed in Hela cells. The secretion, stability and VWF-cleaving activity of the mutants under static and flow conditions were examined. RESULTS: The mutations impaired secretion of ADAMTS-13 to apical surface, but not to extracellular matrix of transfected Hela cells. C1213Y and K1256FS also accelerated, whereas W1245del delayed, extracellular degradation of the mutants. The mutations also resulted in a moderate decrease in cleaving plasma VWF under static conditions. However, the mutated ADAMTS-13 bound to VWF substrate similarly as the wild-type metalloprotease and remained active in cleaving (UL)VWF under flow conditions. CONCLUSIONS: The CUB-1 domain is critical for ADAMTS-13 secretion and stability upon secretion. ADAMTS-13 deficiency found in TTP patients could be resulted from reduced ADAMTS-13 secretion and, in the case of C1213Y and K1256FS accelerated degradation. W1245del is highly resistant to degradation and active in cleaving VWF.

Testosterone enhances early cardiac remodeling after myocardial infarction, causing rupture and degrading cardiac function.

Cardiac rupture can be fatal after myocardial infarction (MI). Experiments in animals revealed gender differences in rupture rate; however, patient data are controversial. We found a significantly higher rupture rate in testosterone-treated female mice within 1 wk after MI, whereas castration in males significantly reduced rupture. We hypothesized that testosterone may adversely affect remodeling after MI, exaggerating the inflammatory response and increasing cardiac rupture, whereas estrogen may be cardioprotective, attenuating early remodeling and reducing rupture rate. We studied the effect of gender and hormone manipulation on morphological and histological changes during early remodeling after MI in 4-wk-old male and female C57BL/6J mice and how these events could affect cardiac function. Females were randomly divided into 1) sham ovariectomy + placebo (s-ovx + P), 2) s-ovx + testosterone (T), 3) ovx + P, and 4) ovx + T; males were divided into 1) sham castration + P (s-cas + P), 2) s-cas + 17beta-estradiol (E), 3) cas + P, and 4) cas + E. At 6 wk after gonadectomy and hormone manipulation, MI was induced. Mice were randomly killed 1, 2, 4, 7, and 14 days after MI. The left ventricle was weighed and sectioned for evaluation of MI size, infarct expansion index (IEI), and neutrophil infiltration. Transthoracic echocardiography was performed in conscious mice in the 14-day group before organ harvest. Cardiac rupture rate and IEI were significantly higher in testosterone-treated females and noncastrated males than in controls; these effects were accompanied by enhanced neutrophil infiltration and pronounced deterioration of cardiac function and left ventricular dilatation. Ovariectomy in females and estrogen supplementation in males did not confer significant protection from cardiac rupture, IEI, or neutrophil infiltration. We concluded that, in mice, high testosterone levels enhance acute myocardial inflammation, adversely affecting myocardial healing and early remodeling, as indicated by increased cardiac rupture, and possibly causing deterioration of cardiac function after MI, and, conversely, estrogen seems to have no significant protective effect in the acute phase after MI.

Cleavage of ultralarge multimers of von Willebrand factor by C-terminal-truncated mutants of ADAMTS-13 under flow.

A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.

Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers.

Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse.

Cyclooxygenase (COX)-2 is expressed in the heart in animal models of ischemic injury. Recent studies have suggested that COX-2 products are involved in inflammatory cell infiltration and fibroblast proliferation in the heart. Using a mouse model, we questioned whether 1). myocardial infarction (MI) in vivo induces COX-2 expression chronically, and 2). COX-2 inhibition reduces collagen content and improves cardiac function in mice with MI. MI was produced by ligation of the left anterior descending coronary artery in mice. Two days later, mice were treated with 3 mg/kg NS-398, a selective COX-2 inhibitor, or vehicle in drinking water for 2 wk. After the treatment period, mice were subjected to two-dimensional M-mode echocardiography to determine cardiac function. Hearts were then analyzed for determination of infarct size, interstitial collagen content, brain natriuretic peptide (BNP) mRNA, myocyte cross-sectional area, and immunohistochemical staining for transforming growth factor (TGF)-beta and COX-2. COX-2 protein, detected by immunohistochemistry, was increased in MI versus sham hearts. MI resulted in increased left ventricular systolic and diastolic dimension and decreased ejection fraction, fractional shortening, and cardiac output. NS-398 treatment partly reversed these detrimental changes. Myocyte cross-sectional area, a measure of hypertrophy, was decreased by 30% in the NS-398 versus vehicle group, but there was no effect on BNP mRNA. The interstitial collagen fraction increased from 5.4 +/- 0.4% in sham hearts to 10.4 +/- 0.9% in MI hearts and was decreased to 7.9 +/- 0.6% in NS-398-treated hearts. A second COX-2 inhibitor, rofecoxib (MK-0966), also decreased myocyte cross-sectional area and interstitial collagen fraction. TGF-beta, a key regulator of collagen synthesis, was increased in MI hearts. NS-398 treatment reduced TGF-beta immunostaining by 40%. NS-398 treatment had no effect on infarct size. These results suggest that COX-2 products contribute to cardiac remodeling and functional deficits after MI. Thus selected inhibition of COX-2 may be a therapeutic target for reducing myocyte damage after MI.