Excess Linoleic Acid Increases Collagen I/III Ratio and "Stiffens" the Heart Muscle Following High Fat Diets.

Controversy exists on the benefits versus harms of n-6 polyunsaturated fatty acids (n-6 PUFA). Although n-6 PUFA demonstrates anti-atherosclerotic properties, survival following cardiac remodeling may be compromised. We hypothesized that n-6 PUFA like linoleic acid (LA) or other downstream PUFAs like γ-linolenic acid or arachidonic acid alter the transforming growth factor-ß (TGFß)-collagen axis in the heart. Excess dietary LA increased the collagen I/III ratio in the mouse myocardium, leading to cardiac "stiffening" characterized by impaired transmitral flow indicative of early diastolic dysfunction within 5 weeks. In vitro, LA under TGFß1 stimulation increased collagen I and lysyl oxidase (LOX), the enzyme that cross-links soluble collagen resulting in deposited collagen. Overexpression of fatty acid desaturase 2 (fads2), which metabolizes LA to downstream PUFAs, reduced collagen deposits, LOX maturation, and activity with LA, whereas overexpressing fads1, unrelated to LA desaturation, did not. Furthermore, fads2 knockdown by RNAi elevated LOX activity and collagen deposits in fibroblasts with LA but not oleic acid, implying a buildup of LA for aggravating such pro-fibrotic effects. As direct incubation with γ-linolenic acid or arachidonic acid also attenuated collagen deposits and LOX activity, we concluded that LA itself, independent of other downstream PUFAs, promotes the pro-fibrotic effects of n-6 PUFA. Overall, these results attempt to reconcile opposing views of n-6 PUFA on the cardiovascular system and present evidence supporting a cardiac muscle-specific effect of n-6 PUFAs. Therefore, aggravation of the collagen I/III ratio and cardiac stiffening by excess n-6 PUFA represent a novel pathway of cardiac lipotoxicity caused by high n-6 PUFA diets.

Partial deletion of ROCK2 protects mice from high-fat diet-induced cardiac insulin resistance and contractile dysfunction.

Obesity is associated with cardiac insulin resistance and contractile dysfunction, which contribute to the development of heart failure. The RhoA-Rho kinase (ROCK) pathway has been reported to modulate insulin resistance, but whether it is implicated in obesity-induced cardiac dysfunction is not known. To test this, wild-type (WT) and ROCK2(+/-) mice were fed normal chow or a high-fat diet (HFD) for 17 wk. Whole body insulin resistance, determined by an insulin tolerance test, was observed in HFD-WT, but not HFD-ROCK2(+/-), mice. The echocardiographically determined myocardial performance index, a measure of global systolic and diastolic function, was significantly increased in HFD-WT mice, indicating a deterioration of cardiac function. However, no change in myocardial performance index was found in hearts from HFD-ROCK2(+/-) mice. Speckle-tracking-based strain echocardiography also revealed regional impairment in left ventricular wall motion in hearts from HFD-WT, but not HFD-ROCK2(+/-), mice. Activity of ROCK1 and ROCK2 was significantly increased in hearts from HFD-WT mice, and GLUT4 expression was significantly reduced. Insulin-induced phosphorylation of insulin receptor substrate (IRS) Tyr(612), Akt, and AS160 was also impaired in these hearts, while Ser(307) phosphorylation of IRS was increased. In contrast, the increase in ROCK2, but not ROCK1, activity was prevented in hearts from HFD-ROCK2(+/-) mice, and cardiac levels of TNFα were reduced. This was associated with normalization of IRS phosphorylation, downstream insulin signaling, and GLUT4 expression. These data suggest that increased activation of ROCK2 contributes to obesity-induced cardiac dysfunction and insulin resistance and that inhibition of ROCK2 may constitute a novel approach to treat this condition.

Selective alpha(1)-adrenoceptor blockade prevents fructose-induced hypertension.

The purpose of this study was to investigate the effect of chronic treatment with prazosin, a selective α1-adrenoceptor antagonist, on the development of hypertension in fructose-fed rats (FFR). High-fructose feeding and treatment with prazosin (1 mg/kg/day via drinking water) were initiated simultaneously in male Wistar rats. Systolic blood pressure, fasted plasma parameters, insulin sensitivity, plasma norepinephrine (NE), uric acid, and angiotensin II (Ang II) were determined following 9 weeks of treatment. FFR exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia, and hypertension, as well as elevations in plasma NE and Ang II levels. Treatment with prazosin prevented the rise in blood pressure without affecting insulin levels, insulin sensitivity, uric acid, or Ang II levels, while normalizing plasma NE levels in FFR. These data suggest that over-activation of the sympathetic nervous system, specifically α1-adrenoceptors, contributes to the development of fructose-induced hypertension, however, this over-activation does not appear to an initial, precipitating event in FFR.

Sex-Specific Acute Cerebrovascular Responses to Photothrombotic Stroke in Mice.

Mechanisms underlying cerebrovascular stroke outcomes are poorly understood, and the effects of biological sex on cerebrovascular regulation post-stroke have yet to be fully comprehended. Here, we explore the overlapping roles of gonadal sex hormones and rho-kinase (ROCK), two important modulators of cerebrovascular tone, on the acute cerebrovascular response to photothrombotic (PT) focal ischemia in mice. Male mice were gonadectomized and female mice were ovariectomized to remove gonadal hormones, whereas control ("intact") animals received a sham surgery prior to stroke induction. Intact wild-type (WT) males showed a delayed drop in cerebral blood flow (CBF) compared with intact WT females, whereby maximal CBF drop was observed 48 h following stroke. Gonadectomy in males did not alter this response. However, ovariectomy in WT females produced a "male-like" phenotype. Intact Rock2+/- males also showed the same phenotypic response, which was not altered by gonadectomy. Alternatively, intact Rock2+/- females showed a significant difference in CBF values compared with intact WT females, displaying higher CBF values immediately post-stroke and showing a maximal CBF drop 48 h post-stroke. This pattern was not altered by ovariectomy. Altogether, these data illustrate sex differences in acute CBF responses to PT stroke, which seem to involve gonadal female sex hormones and ROCK2. Overall, this study provides a framework for exploring sex differences in acute CBF responses to focal ischemic stroke in mice.

Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCß2.

We previously reported that acute inhibition of the RhoA/Rho kinase (ROCK) pathway normalized contractile function of diabetic rat hearts, but the underlying mechanism is unclear. Protein kinase C (PKC) ß(2) has been proposed to play a major role in diabetic cardiomyopathy at least in part by increasing oxidative stress. Further evidence suggests that PKC positively regulates RhoA expression through induction of inducible nitric oxide synthase (iNOS) in diabetes. However, in preliminary studies, we found that inhibition of ROCK itself reduced RhoA expression in diabetic hearts. We hypothesized that there is an interaction between RhoA/ROCK and PKCß(2) in the form of a positive feedback loop that sustains their activation and the production of reactive oxygen species (ROS). This was investigated in cardiomyocytes isolated from diabetic and control rat hearts, incubated with or without cytochalasin D or inhibitors of ROCK, RhoA, PKCß(2), or iNOS. Inhibition of RhoA and ROCK markedly attenuated the diabetes-induced increases in PKCß(2) activity and iNOS and RhoA expression in diabetic cardiomyocytes, while having no effect in control cells. Inhibition of PKCß(2) and iNOS also normalized RhoA expression and ROCK overactivation, whereas iNOS inhibition reversed the increase in PKCß(2) activity. Each of these treatments also normalized the diabetes-induced increase in production of ROS. Actin cytoskeleton disruption attenuated the increased expression and/or activity of all of these targets in diabetic cardiomyocytes. These data suggest that, in the diabetic heart, the RhoA/ROCK pathway contributes to contractile dysfunction at least in part by sustaining PKCß(2) activation and ROS production via a positive feedback loop that requires an intact cytoskeleton.

Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.

The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa+ SCA-1+ cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.

The ischemic metabolite lysophosphatidylcholine increases rat coronary arterial tone by endothelium-dependent mechanisms.

Lysophosphatidylcholine (LPC), a hydrolysis product of phospholipid degradation, accumulates in the ischemic myocardium. Using isolated hearts or rat coronary septal arteries, we tested the impact of LPC in modulating basal function or the responses to vasoactive agents. Sustained perfusion of hearts with LPC augmented coronary perfusion pressure (CPP) and reduced left ventricular developed pressure (LVDP). By mechanisms that have yet to be identified, these effects on CPP and LVDP were exaggerated when LPC was removed from the perfusate. Although LPC (or its washout) had no direct effect on vascular tone in the isolated coronary artery, it selectively potentiated the receptor-coupled vasoconstrictor response to U-46619, a thromboxane A(2) mimetic. Interestingly, when LPC was washed out, the potentiation to U-46619 was even more pronounced. Both the immediate and residual effects of LPC were endothelium-dependent. EDHF was likely the sole mediator responsible for the direct effects of LPC on U-46619-vasoconstriction, whereas the augmented vasoconstrictor responses following LPC washout may in part be related to an increase in ET-1, and a striking reduction in the bioavailability of NO. Our data suggest that in addition to reducing the accumulation of LPC to prevent ischemia-reperfusion (I/R) damage, efforts targeting an improved endothelium-dependent regulation of vascular tone could be an attractive approach to limit the cardiac damage induced by I/R.

Chronic etanercept treatment prevents the development of hypertension in fructose-fed rats.

The purpose of this study was to investigate the effect of chronic treatment with etanercept (a soluble recombinant fusion protein consisting of the extracellular ligand-binding domain of tumor necrosis factor receptor type 2) on the development of hypertension in fructose-fed rats (FFR). High fructose feeding and treatment with etanercept (0.3 mg/kg, three times per week) was initiated simultaneously in male Wistar rats. Systolic blood pressure, fasted plasma parameters, insulin sensitivity, vascular reactivity, plasma angiotensin II (Ang II), and norepinephrine were determined following 9 weeks of treatment. FFR exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia, endothelial dysfunction, and hypertension. Treatment with etanercept prevented the rise in blood pressure without affecting insulin levels, insulin sensitivity, triglycerides, or Ang II levels in FFR. Etanercept treatment improved acetylcholine-induced relaxation and normalized endothelial nitric oxide synthase expression in aortas from FFR. The results of this study suggest that treatment with etanercept prevented the development of hypertension by improving vascular function and restoring endothelial nitric oxide synthase expression in FFR.

Role of inducible nitric oxide synthase in induction of RhoA expression in hearts from diabetic rats.

AIMS: Recent studies from our laboratory demonstrated that increased expression of the small GTP-binding protein RhoA and activation of the RhoA/rho kinase (ROCK) pathway play an important role in the contractile dysfunction associated with diabetic cardiomyopathy in hearts from streptozotocin (STZ)-induced diabetic rats. Nitric oxide (NO) has been reported to be a positive regulator of RhoA expression in vascular smooth muscle, and we have previously found that the expression of inducible NO synthase (iNOS) is increased in hearts from STZ-diabetic rats. Therefore, in this study, we investigated the hypothesis that induction of iNOS positively regulates RhoA expression in diabetic rat hearts. METHODS AND RESULTS: To determine whether NO and iNOS could increase RhoA expression in the heart, cardiomyocytes from non-diabetic rats were cultured in the presence of the NO donor sodium nitroprusside (SNP) or lipopolysaccharide (LPS) in the absence and presence of the selective iNOS inhibitor, N(6)-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL). In a second study, 1 week after induction of diabetes with STZ, rats were treated with L-NIL (3 mg/kg/day) for 8 more weeks to determine the effect of iNOS inhibition in vivo on RhoA expression and cardiac contractile function. Expression of iNOS was elevated in cardiomyocytes isolated from diabetic rat hearts. Both SNP and LPS increased RhoA expression in non-diabetic cardiomyocytes. The LPS-induced elevation in RhoA expression was accompanied by an increase in iNOS expression and prevented by L-NIL. Treatment of diabetic rats with L-NIL led to a significant improvement in left ventricular developed pressure and rates of contraction and relaxation concomitant with normalization of total cardiac nitrite levels, RhoA expression, and phosphorylation of the ROCK targets LIM (Lin-11, Isl-1, Mec-3) kinase and ezrin/radixin/moesin. CONCLUSION: These data suggest that iNOS is involved in the increased expression of RhoA in diabetic hearts and that one of the mechanisms by which iNOS inhibition improves cardiac function is by preventing the upregulation of RhoA and its availability for activation.

ROCK2 promotes ryanodine receptor phosphorylation and arrhythmic calcium release in diabetic cardiomyocytes.

BACKGROUND: Diabetes is associated with an increased risk of heart failure, cardiac arrhythmias and sudden cardiac death. We previously showed that ROCK2 expression is elevated in diabetic rat hearts, and that ROCK inhibition acutely improves their contractile function. In the present study we investigated whether inhibition of ROCK or partial deletion of ROCK2 improves impaired Ca2+ handling in the diabetic heart. METHODS: Contractile properties and Ca2+ transients were measured before and after treatment with the ROCK inhibitor Y-27632 (1 µM) in fluo-4-loaded cardiomyocytes isolated from streptozotocin (STZ)-diabetic or non-diabetic rats. Cardiac function was determined in vivo, and contractile properties and Ca2+ transients also measured in cardiomyocytes from non-diabetic and STZ-diabetic wild-type (WT) and ROCK2+/- mice. RESULTS: ROCK inhibition improved some parameters of contractile function and Ca2+ handling in cardiomyocytes from diabetic rat hearts. In addition, ROCK inhibition attenuated the diabetes-induced delayed aftercontractions (DACs) and associated irregular Ca2+ transients induced by increased [Ca2+]o. Although no overt cardiac dysfunction was detected in diabetic WT mice, cardiomyocytes from these mice also developed arrhythmic Ca2+ transients in response to increased [Ca2+]. These were attenuated in cardiomyocytes from diabetic ROCK2+/- mice, in association with decreased diastolic Ca2+ leak and with reduction of the diabetes-induced increased phosphorylation of both CaMKII and the ryanodine receptor (RyR). CONCLUSIONS: These data suggest that ROCK2 contributes to diabetes-induced impaired cardiac Ca2+ homeostasis, at least in part by promoting CaMKII-mediated phosphorylation of RyR. This may have important clinical implications for the treatment of the increased incidence of dysrhythmias in diabetes.

Acute inhibition of Rho-kinase improves cardiac contractile function in streptozotocin-diabetic rats.

OBJECTIVE: The purpose of the present study was to determine whether increased activation of the RhoA/Rho-kinase (ROCK) pathway occurs in diabetic cardiomyopathy and whether acute inhibition of this pathway improves contractile function of the diabetic heart. METHODS: Male Wistar rats were made diabetic with streptozotocin. Twelve to fourteen weeks later, the effects of acute administration of the ROCK inhibitors Y-27632 and H-1152 on cardiac contractile function were measured both in vitro, in isolated working hearts, and in vivo, using echocardiography. Changes in the expression and activity of RhoA, and the effect of ROCK inhibition on changes in the phosphorylation of the downstream target of ROCK, LIM kinase 2, and on actin polymerization in diabetic hearts were also determined. RESULTS: Perfusion of isolated working hearts from diabetic rats with Y-27632 or H-1152 acutely improved left ventricle developed pressure and the rates of contraction and relaxation. Acute administration of H-1152 also significantly improved the percent fraction shortening, an index of left ventricle contractility, in vivo in diabetic rats. The expression and activity of RhoA in cardiomyocytes from diabetic rats were significantly increased, as was the phosphorylation of LIM kinase 2. This was associated with an increase in actin polymerization (the F-actin to G-actin ratio). Both the increase in LIM kinase 2 phosphorylation and actin polymerization were attenuated by ROCK inhibition. CONCLUSIONS: These data suggest that activation of the RhoA/ROCK signaling pathway plays a critical role in the development of diabetic cardiomyopathy, and that ROCK is an excellent therapeutic target in the treatment of this condition.

Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to alpha-adrenoceptor stimulation.

Protein kinase C (PKC) may contribute to enhanced contractile responses of arteries from streptozotocin-diabetic rats to stimulation of G-protein coupled receptors. This was investigated by comparing the effects of PKC inhibitors on contractile responses of mesenteric arteries from diabetic and age-matched control rats to noradrenaline (NA) and endothelin-1 (ET-1). The effects of NA and ET-1 on the distribution of three isoforms of PKC implicated in contraction were also determined. In addition, the effect of NA on phosphorylation of CPI-17, a substrate for PKC, was investigated. Contractile responses of endothelium-denuded arteries from diabetic rats to NA were enhanced, but were normalized by PKC inhibition. In contrast, contractile responses to ET-1 were not significantly different, and were blocked to a similar extent by PKC inhibition, in arteries from control and diabetic rats.NA produced only a small increase in particulate levels of PKCepsilon in control arteries (to 125+/-8% of levels in untreated arteries), but a significant increase in particulate PKCalpha (to 190+/-22%) and a much greater increase in particulate PKCepsilon (to 230+/-19%) in arteries from diabetic rats. ET-1 increased particulate PKCalpha and epsilon to a similar extent in arteries from control and diabetic rats.NA significantly enhanced CPI-17 phosphorylation from a basal level of 22+/-10 to 71+/-7% of total in arteries from diabetic rats, and this was prevented by PKC inhibition. NA had no detectable effect on CPI-17 phosphorylation in arteries from control rats. These data suggest that NA-induced activation of PKC and CPI-17, its downstream target, is selectively enhanced in arteries from diabetic rats, and mediates the enhanced contractile responses to this agonist.

Age-dependent changes in blood pressure and arterial reactivity in obese Zucker rats.

The purpose of the present investigation was to determine whether there is an association between changes in arterial reactivity to vasoactive agents and the development of hypertension in obese Zucker rats. At 20 weeks of age, obese rats were mildly hypotensive compared to their lean littermate controls. Maximum contractile responses of endothelium-intact mesenteric arteries from these rats to noradrenaline, endothelin-1 and KCl were depressed, although there was no change in relaxation to acetylcholine. By 32 weeks of age, obese rats had developed hypertension compared to their lean littermate controls. Maximum contractile responses of mesenteric arteries from 32-week-old obese rats to noradrenaline and endothelin-1 were no longer significantly different than control, although contractile responses to KCl remained depressed. In addition, there was a small increase in sensitivity to endothelin-1, while endothelium-dependent relaxation to acetylcholine was impaired. In contrast, there were no changes in contractile responses of endothelium-intact aortas from either 20- or 32-week-old obese rats to noradrenaline, endothelin-1 or KCl, while endothelium-dependent relaxation of this artery to acetylcholine was slightly enhanced at both ages. Therefore, changes in the reactivity of the mesenteric artery but not the aorta from obese Zucker rats parallel changes in blood pressure in these animals.

Complex regulation of PKCß2 and PDK-1/AKT by ROCK2 in diabetic heart.

OBJECTIVES: The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCß2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCß2, and to determine if their interaction affects PDK-1/Akt signaling. METHODS: Regulation by ROCK of PKCß2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCß2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCß2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy. RESULTS: Expression of ROCK2 and expression and phosphorylation of PKCß2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCß2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCß2 T641 phosphorylation, and this was associated with attenuation of PKCß2 activity. PKCß2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts. CONCLUSION: This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCß2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes.

A pragmatic, multicentre, randomised controlled trial comparing stapled haemorrhoidopexy to traditional excisional surgery for haemorrhoidal disease (eTHoS): study protocol for a randomised controlled trial.

BACKGROUND: Current interventions for haemorrhoidal disease include traditional haemorrhoidectomy (TH) and stapled haemorrhoidopexy (SH) surgery. However, uncertainty remains as to how they compare from a clinical, quality of life (QoL) and economic perspective. The study is therefore designed to determine whether SH is more effective and more cost-effective, compared with TH. METHODS/DESIGN: eTHoS (either Traditional Haemorrhoidectomy or Stapled Haemorrhoidopexy for Haemorrhoidal Disease) is a pragmatic, multicentre, randomised controlled trial. Currently, 29 secondary care centres are open to recruitment. Patients, aged 18 year or older, with circumferential haemorrhoids grade II to IV, are eligible to take part. The primary clinical and economic outcomes are QoL profile (area under the curve derived from the EuroQol Group's 5 Dimension Health Status Questionnaire (EQ-5D) at all assessment points) and incremental cost per quality adjusted life year (QALY) based on the responses to the EQ-5D at 24 months. The secondary outcomes include a comparison of the SF-36 scores, pain and symptoms sub-domains, disease recurrence, complication rates and direct and indirect costs to the National Health Service (NHS). A sample size of n =338 per group has been calculated to provide 90% power to detect a difference in the mean area under the curve (AUC) of 0.25 standard deviations derived from EQ-5D score measurements, with a two-sided significance level of 5%. Allowing for non-response, 400 participants will be randomised per group. Randomisation will utilise a minimisation algorithm that incorporates centre, grade of haemorrhoidal disease, baseline EQ-5D score and gender. Blinding of participants and outcome assessors is not attempted. DISCUSSION: This is one of the largest trials of its kind. In the United Kingdom alone, 29,000 operations for haemorrhoidal disease are done annually. The trial is therefore designed to give robust evidence on which clinicians and health service managers can base management decisions and, more importantly, patients can make informed choices. TRIAL REGISTRATION: Current Controlled Trials ISRCTN80061723 (assigned 8 March 2010).

Contribution of Rho kinase to blood pressure elevation and vasoconstrictor responsiveness in type 2 diabetic Goto-Kakizaki rats.

OBJECTIVES: The RhoA-Rho kinase (ROCK) pathway contributes to a number of diabetic complications including cardiomyopathy and nephropathy. In this study, we investigated whether it contributes to elevated blood pressure and vascular contractile dysfunction in type 2 diabetes. METHODS: Blood pressure was measured in Goto-Kakizaki rats, a nonobese model of type 2 diabetes, before and after treatment with the ROCK inhibitor fasudil. Vasoconstrictor responsiveness in the absence and presence of ROCK inhibitors as well as ROCK pathway activity was measured in isolated mesenteric resistance vessels from these animals. RESULTS: Blood pressure was elevated in diabetic rats compared with age-matched Wistar controls, and was normalized by treatment with fasudil. Contractile responses of mesenteric arteries from diabetic rats to phenylephrine and U-46619, as well as relaxant responses to acetylcholine, were unaltered. However, vasoconstrictor responses were more sensitive to ROCK inhibition with either Y-27632 or H-1152 than were responses of control arteries. No differences were found in expression of RhoA, ROCK1, or ROCK2 or in basal ROCK activity between arteries from control and diabetic rats. U-46619 produced a similar magnitude of increase in ROCK activity that was completely blocked by H-1152 in arteries from both groups of animals. CONCLUSION: These data suggest that ROCK contributes to the increase in blood pressure in type 2 diabetic Goto-Kakizaki rats, and that vasoconstrictor responses of small mesenteric arteries from these animals are more dependent on ROCK than are responses of control arteries.

Fat Embolism Syndrome following minor trauma in Duchenne muscular dystrophy.

We describe five patients with Duchenne muscular dystrophy who presented with acute neurologic and respiratory symptoms following minor trauma. Four of the five deteriorated rapidly and died within 36 h after falling. X-rays for fractures were negative. Four of the five patients were taking corticosteroids daily. All five patients fulfilled the clinical criteria for Fat Embolism Syndrome. Autopsy findings were consistent with fat embolism in two cases. Fat Embolism Syndrome needs to be considered in patients with Duchenne muscular dystrophy following minor trauma even without fractures. Early recognition of Fat Embolism Syndrome and aggressive resuscitation are important to improve survival. This report serves as an important reminder that seatbelts need to be used at all times.

GPCR agonist-induced transactivation of the EGFR upregulates MLC II expression and promotes hypertension in insulin-resistant rats.

AIMS: The presence of metabolic abnormalities such as insulin resistance and elevated levels of various vasoconstrictor G-protein-coupled receptor (GPCR) agonists contributes to the development of hypertension. Recent studies have suggested a link between disease progression and activation of growth factor receptor signalling pathways such as the epidermal growth factor receptor (EGFR) by matrix metalloproteinases (MMPs). We hypothesized that excessive stimulation of GPCRs such as alpha(1)-adrenergic receptors activates MMP-dependent EGFR transactivation and contributes to the development of hypertension by promoting increased synthesis of contractile proteins in vascular smooth muscle (VSM). METHODS AND RESULTS: We tested this concept in experiments using insulin-resistant VSM cells (VSMCs) and fructose hypertensive rats (FHRs), a model of acquired systolic hypertension and insulin resistance. We found that insulin resistance and agonist stimulation increased the expression and activity of MMPs (MMP-2 and MMP-7), the EGFR, contractile proteins such as myosin light chain kinase and MLC II, and their transcriptional activators including P90 ribosomal kinase (P90RSK) and serum response factor, possibly via the activation of extracellular signal-regulated kinase (ERK1/2) in VSMCs. Further, in insulin-resistant VSMCs and arteries from FHRs, disruption of MMP-EGFR signalling either by a pharmacological or small interfering RNA approach normalized the increased expression and activity of contractile proteins and their transcriptional activators and prevented the development of hypertension in FHRs. CONCLUSION: Our data suggest that the MMP-EGFR pathway could be a potential target in the treatment of hypertension in insulin resistance and/or hyperglycaemic conditions such as type 2 diabetes.