Loss-of-function G6PD variant moderated high fat diet-induced obesity, adipocyte hypertrophy, and fatty liver in male rats.

Obesity is a major risk factor for liver and cardiovascular diseases. However, obesity-driven mechanisms that contribute to the pathogenesis of multiple organ diseases are still obscure and treatment is inadequate. We hypothesized that increased glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme in the pentose shunt, is critical in evoking metabolic reprogramming in multiple organs and is a significant contributor to the pathogenesis of liver and cardiovascular diseases. G6PD is induced by carbohydrate-rich diet and insulin. Long-term (8 months) high-fat diet (HFD) feeding increased body weight and elicited metabolic reprogramming in visceral fat, liver, and aorta, of the wild-type rats. In addition, HFD increased inflammatory chemokines in visceral fat. Interestingly, CRISPR-edited loss-of-function Mediterranean G6PD variant (G6PDS188F) rats, which mimic human polymorphism, moderated HFD-induced weight gain and metabolic reprogramming in visceral fat, liver, and aorta. The G6PDS188F variant prevented HFD-induced CCL7 and adipocyte hypertrophy. Furthermore, the G6PDS188F variant increased Magel2 - a gene encoding circadian clock-related protein that suppresses obesity associated with Prader-Willi syndrome - and reduced HFD-induced non-alcoholic fatty liver. Additionally, the G6PDS188F variant reduced aging-induced aortic stiffening. Our findings suggest G6PD is a regulator of HFD-induced obesity, adipocyte hypertrophy, and fatty liver.

Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion.

Background: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. Methods: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. Results: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. Conclusion: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway.

G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases.

We aimed to determine 1) the mechanism(s) that enables glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)-driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs, and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC-restricted genes. G6PD knockdown or inhibition decreased (48.5%) histone deacetylase (HDAC) activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control Sprague-Dawley (SD) rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4 mo increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate that G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.NEW & NOTEWORTHY This study gives detailed mechanistic insight about the regulation of smooth muscle cell (SMC) phenotype by metabolic reprogramming and glucose-6-phosphate dehydrogenase (G6PD) in diabetes and metabolic syndrome. We demonstrate that G6PD controls the chromatin modifications by regulating histone deacetylase (HDAC) activity, which deacetylates histone 3-lysine 9 and 27. Notably, inhibition of G6PD decreases HDAC activity and enriches H3K27ac on myocardin gene promoter to enhance the expression of SMC-restricted genes. Also, we demonstrate for the first time that G6PD inhibitor treatment accentuates metabolic and transcriptomic reprogramming to reduce neointimal formation in coronary artery and large artery elastance in metabolic syndrome rats.

CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant.

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase (G6pd) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PDS188F) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced (P<0.05) in the arteries of G6PDS188F versus WT rats. Intriguingly, G6PDS188F mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-NG-nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PDS188F rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K+ channel proteins and (2) decreased voltage-gated Ca2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-NG-nitroarginine methyl ester and high-fat diet to G6PDS188F mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.

Glucose-6-phosphate dehydrogenase increases Ca2+ currents by interacting with Cav1.2 and reducing intrinsic inactivation of the L-type calcium channel.

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

Pathophysiology of chronic peripheral ischemia: new perspectives.

Peripheral arterial disease (PAD) affects individuals particularly over 65 years old in the more advanced countries. Hemodynamic, inflammatory, and oxidative mechanisms interact in the pathophysiological scenario of this chronic arterial disease. We discuss the hemodynamic, muscle tissue, and oxidative stress (OxS) conditions related to chronic ischemia of the peripheral arteries. This review summarizes the results of evaluating both metabolic and oxidative markers, and also therapy to counteract OxS. In conclusion, we believe different pathways should be highlighted to discover new drugs to treat patients suffering from PAD.

Comparison of Cardiovascular Benefits of Bariatric Surgery and Abdominal Lipectomy.

PURPOSE OF REVIEW: The purpose of this review is to examine recent evidence supporting effectiveness of bariatric surgery and abdominal lipectomy as interventional strategies aimed at reduction in incidence of cardiovascular disease (CVD) and related morbidity and mortality in obese and metabolic syndrome patients. RECENT FINDINGS: While several studies show reduction in CVD risk factors in patients who have undergone both the Roux-en-Y gastric bypass and sleeve gastrectomy, very few demonstrate actual improvements in cardiovascular function, or a decrease in CVD events or CVD-related mortality. Consequently, the cardiovascular benefits of the less invasive sleeve gastrectomy in comparison to the gastric bypass are also unclear. Striking new data on large patient samples demonstrate significant positive correlation between gastric bypass and CVD risk factor reduction only in patients who are diabetic or > 50 years of age at the time of surgery, with no significant differences in non-diabetic and younger patients and with significant side effects. On the other hand, a markedly less invasive removal of abdominal subcutaneous adipose tissue via lipectomy consistently and significantly improved CVD risk factors as well as cardiovascular function in the very few studies available. Overall, neither the potential nor the definitive cardiovascular benefits of either of the commonly used bariatric surgical or the various lipectomy procedures have been adequately explored. Future basic science and clinical studies have the opportunity to understand the mechanisms and long-term consequences of both approaches and develop personalized approaches with higher benefit to side effect ratios.

20-HETE in the regulation of vascular and cardiac function.

20-HETE, the ω-hydroxylation product of arachidonic acid catalyzed by enzymes of the cytochrome P450 (CYP) 4A and 4F gene families, is a bioactive lipid mediator with potent effects on the vasculature including stimulation of smooth muscle cell contractility, migration and proliferation as well as activation of endothelial cell dysfunction and inflammation. Clinical studies have shown elevated levels of plasma and urinary 20-HETE in human diseases and conditions such as hypertension, obesity and metabolic syndrome, myocardial infarction, stroke, and chronic kidney diseases. Studies of polymorphic associations also suggest an important role for 20-HETE in hypertension, stroke and myocardial infarction. Animal models of increased 20-HETE production are hypertensive and are more susceptible to cardiovascular injury. The current review summarizes recent findings that focus on the role of 20-HETE in the regulation of vascular and cardiac function and its contribution to the pathology of vascular and cardiac diseases.

Elevated 20-HETE in metabolic syndrome regulates arterial stiffness and systolic hypertension via MMP12 activation.

Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182 ±â€¯3 mmHg JCR, 145 ±â€¯3 mmHg JCR + 20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Cardiovascular function in male and female JCR:LA-cp rats: effect of high-fat/high-sucrose diet.

Thirty percent of the world population is diagnosed with metabolic syndrome. High-fat/high-sucrose (HF/HS) diet (Western diet) correlates with metabolic syndrome prevalence. We characterized effects of the HF/HS diet on vascular (arterial stiffness, vasoreactivity, and coronary collateral development) and cardiac (echocardiography) function, oxidative stress, and inflammation in a rat model of metabolic syndrome (JCR rats). Furthermore, we determined whether male versus female animals were affected differentially by the Western diet. Cardiovascular function in JCR male rats was impaired versus normal Sprague-Dawley (SD) rats. HF/HS diet compromised cardiovascular (dys)function in JCR but not SD male rats. In contrast, cardiovascular function was minimally impaired in JCR female rats on normal chow. However, cardiovascular function in JCR female rats on the HF/HS diet deteriorated to levels comparable to JCR male rats on the HF/HS diet. Similarly, oxidative stress was markedly increased in male but not female JCR rats on normal chow but was equally exacerbated by the HF/HS diet in male and female JCR rats. These results indicate that the Western diet enhances oxidative stress and cardiovascular dysfunction in metabolic syndrome and eliminates the protective effect of female sex on cardiovascular function, implying that both males and females with metabolic syndrome are at equal risk for cardiovascular disease.NEW & NOTEWORTHY Western diet abolished protective effect of sex against cardiovascular disease (CVD) development in premenopausal animals with metabolic syndrome. Western diet accelerates progression of CVD in male and female animals with preexisting metabolic syndrome but not normal animals. Exacerbation of baseline oxidative stress correlates with accelerated progression of CVD in metabolic syndrome animals on Western diet.

Can microRNAs be Biomarkers or Targets for Therapy of Ischemic Coronary Artery Disease in Metabolic Syndrome?

BACKGROUND: Due to their exceptional stability in the circulation, microRNAs (miRs) are being identified as promising biomarkers. On the other hand, their propensity to regulate networks of functionally closely related genes and relative ease of delivery makes them attractive targets for therapy. However, neither application is without challenges, especially as it applies to ischemic coronary artery disease (CAD). OBJECTIVE: This review will: 1) describe miRs which have been most consistently found to be associated with the most common manifestations of CAD, including atherosclerosis, angina pectoris, myocardial infarction and myocardial reperfusion through arteriogenesis, 2) emphasize those miRs which are also altered in metabolic syndrome and its component pathologies, 3) discuss challenges which currently prevent clinical application related to inconsistencies between findings in cell culture, animal models and among human studies, as well as technical challenges, and 4) offer some suggestions towards resolutions of these discrepancies. CONCLUSION: While miRs can be used as reliable biomarkers for myocardial infarction, their use as biomarkers for other forms of ischemic CAD, as well as therapy for CAD await further investigation.

Elevated 20-HETE impairs coronary collateral growth in metabolic syndrome via endothelial dysfunction.

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS.NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE.

Mechanisms of Comorbidities Associated With the Metabolic Syndrome: Insights from the JCR:LA-cp Corpulent Rat Strain.

Obesity and its metabolic complications have emerged as the epidemic of the new millennia. The use of obese rodent models continues to be a productive component of efforts to understand the concomitant metabolic complications of this disease. In 1978, the JCR:LA-cp rat model was developed with an autosomal recessive corpulent (cp) trait resulting from a premature stop codon in the extracellular domain of the leptin receptor. Rats that are heterozygous for the cp trait are lean-prone, while those that are homozygous (cp/cp) spontaneously display the pathophysiology of obesity as well as a metabolic syndrome (MetS)-like phenotype. Over the years, there have been formidable scientific contributions that have originated from this rat model, much of which has been reviewed extensively up to 2008. The premise of these earlier studies focused on characterizing the pathophysiology of MetS-like phenotype that was spontaneously apparent in this model. The purpose of this review is to highlight areas of recent advancement made possible by this model including; emerging appreciation of the "thrifty gene" hypothesis in the context of obesity, the concept of how chronic inflammation may drive obesogenesis, the impact of acute forms of inflammation to the brain and periphery during chronic obesity, the role of dysfunctional insulin metabolism on lipid metabolism and vascular damage, and the mechanistic basis for altered vascular function as well as novel parallels between the human condition and the female JCR:LA-cp rat as a model for polycystic ovary disease (PCOS).

miR-21-mediated decreased neutrophil apoptosis is a determinant of impaired coronary collateral growth in metabolic syndrome.

Coronary collateral growth (CCG) is impaired in metabolic syndrome. microRNA-21 (miR-21) is a proproliferative and antiapoptotic miR, which we showed to be elevated in metabolic syndrome. Here we investigate whether impaired CCG in metabolic syndrome involved miR-21-mediated aberrant apoptosis. Normal Sprague-Dawley (SD) and metabolic syndrome [J. C. Russel (JCR)] rats underwent transient, repetitive coronary artery occlusion [repetitive ischemia (RI)]. Antiapoptotic Bcl-2, phospho-Bad, and Bcl-2/Bax dimers were increased on days 6 and 9 RI, and proapoptotic Bax and Bax/Bax dimers and cytochrome-c release concurrently decreased in JCR versus SD rats. Active caspases were decreased in JCR versus SD rats (~50%). Neutrophils increased transiently on day 3 RI in the collateral-dependent zone of SD rats but remained elevated in JCR rats, paralleling miR-21 expression. miR-21 downregulation by anti-miR-21 induced neutrophil apoptosis and decreased Bcl-2 and Bcl-2/Bax dimers (~75%) while increasing Bax/Bax dimers, cytochrome-c release, and caspase activation (~70, 400, and 400%). Anti-miR-21 also improved CCG in JCR rats (~60%). Preventing neutrophil infiltration with blocking antibodies resulted in equivalent CCG recovery, confirming a major role for deregulated neutrophil apoptosis in CCG impairment. Neutrophil and miR-21-dependent CCG inhibition was in significant part mediated by increased oxidative stress. We conclude that neutrophil apoptosis is integral to normal CCG and that inappropriate prolonged miR-21-mediated survival of neutrophils plays a major role in impaired CCG, in part via oxidative stress generation.

Role of MMP2 and MMP9 in TRPV4-induced lung injury.

Ca(2+) entry through transient receptor potential vanilloid 4 (TRPV4) results in swelling, blebbing, and detachment of the epithelium and capillary endothelium in the intact lung. Subsequently, increased permeability of the septal barrier and alveolar flooding ensue. In this study, we tested the hypothesis that TRPV4 activation provides a Ca(2+) source necessary for proteolytic disruption of cell-cell or cell-matrix adhesion by matrix metalloproteinases (MMPs) 2 and 9, thus increasing septal barrier permeability. In our study, C57BL/6 or TRPV4(-/-) mouse lungs were perfused with varying doses of the TRPV4 agonist GSK-1016790A (Sigma) and then prepared for Western blot. Lung injury, assessed by increases in lung wet-to-dry weight ratios and total protein levels in the bronchoalveolar lavage fluid, was increased in a dose-dependent fashion in TRPV4(+/+) but not TRPV4(-/-) lungs. In concert with lung injury, we detected increased active MMP2 and MMP9 isoforms, suggesting that TRPV4 can provide the Ca(2+) source necessary for increased MMP2/9 activation. Furthermore, tissue inhibitor of metalloproteinases (TIMP) 2 levels in the TRPV4-injured lungs were decreased, suggesting that TRPV4 activation increases the availability of these active MMPs. We then determined whether MMP2 and MMP9 mediate TRPV4-induced lung injury. Pharmacological blockade (SB-3CT, 1 µM; Sigma) of MMP2 and MMP9 resulted in protection against TRPV4-induced lung injury. We conclude that TRPV4 activation and the subsequent Ca(2+) transient initiates a rapid cascade of events leading to release and activation of the gelatinase MMPs, which then contribute to lung injury.

miR-21 normalizes vascular smooth muscle proliferation and improves coronary collateral growth in metabolic syndrome.

Inadequate cell proliferation is considered a major causative factor for impaired coronary collateral growth (CCG). Proangiogenic growth factors (GFs) stimulate cell proliferation, but their administration does not promote CCG in patients. These GFs are increased in patients with metabolic syndrome and in animal models, where CCG is impaired. Here, we investigated whether excessive cell proliferation underlies impaired CCG in metabolic syndrome. Normal [Sprague-Dawley (SD)] and metabolic syndrome [James C. Russell (JCR)] rats underwent repetitive ischemia (RI; transient, repetitive coronary artery occlusion and myocardial ischemia). We have shown that CCG was maximal at d 9 of RI in SD rats but did not occur in JCR rats. The increase in cell proliferation (PCNA, Ki-67, cyclin A, phospho- cdc2, p21Waf, p27Kip) was transient (∼4-fold, d 3 RI) in SD rats but greater and sustained in JCR rats (∼8- to 6-fold, d 3-9 RI). In JCR rats, this was associated with increased and sustained miR-21 expression and accumulation of proliferating synthetic vascular smooth muscle cells in the lumen of small arterioles, which failed to undergo outward expansion. Administration of anti-miR-21 blocked RI-induced cell proliferation and significantly improved CCG in JCR rats (∼60%). miR-21-dependent excessive cell proliferation in the later stages of collateral remodeling correlates with impaired CCG in metabolic syndrome.

A device for performing automated balloon catheter inflation ischemia studies.

Coronary collateral growth (arteriogenesis) is a physiological adaptive response to transient and repetitive occlusion of major coronary arteries in which small arterioles (native collaterals) with minimal to no blood flow remodel into larger conduit arteries capable of supplying adequate perfusion to tissue distal to the site of occlusion. The ability to reliably and reproducibly mimic transient, repetitive coronary artery occlusion (ischemia) in animal models is critical to the development of therapies to restore coronary collateral development in type II diabetes and the metabolic syndrome. Current animal models for repetitive coronary artery occlusion implement a pneumatic occluder (balloon) that is secured onto the surface of the heart with the suture, which is inflated manually, via a catheter connected to syringe, to effect occlusion of the left anterior descending coronary artery (LAD). This method, although effective, presents complications in terms of reproducibility and practicality. To address these limitations, we have designed a device for automated, transient inflation of balloon catheters in coronary artery occlusion models. This device allows repeated, consistent inflation (to either specified pressure or volume) and the capability for implementing very complex, month-long protocols. This system has significantly increased the reproducibility of coronary collateral growth studies in our laboratory, resulting in a significant decrease in the numbers of animals needed to complete each study while relieving laboratory personnel from the burden of extra working hours and enabling us to continue studies over periods when we previously could not. In this paper, we present all details necessary for construction and operation of the inflator. In addition, all of the components for this device are commercially available and economical (Table S1). It is our hope that the adoption of automated balloon catheter inflation protocols will improve the experimental reliability of transient ischemia studies at many research institutions.

Impaired coronary collateral growth in the metabolic syndrome is in part mediated by matrix metalloproteinase 12-dependent production of endostatin and angiostatin.

OBJECTIVE: We have previously shown that transient coronary artery occlusion stimulated coronary collateral growth (CCG) in healthy (Sprague Dawley) but not in metabolic syndrome (JCR:LA-cp [JCR] ) rats. Here, we sought to determine whether matrix metalloproteinases (MMPs) negatively regulate CCG in the metabolic syndrome via release of endostatin and angiostatin. APPROACH AND RESULTS: Rats underwent transient, repetitive left anterior descending occlusion and resultant myocardial ischemia (RI) for 0 to 10 days. CCG was measured in the collateral-dependent and normal zones using microspheres, MMP activation by Western blot, and endostatin and angiostatin by ELISA on days 0, 3, 6, 9, or 10 of RI. Endostatin and angiostatin were increased in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Increased endostatin and angiostatin correlated with increased MMP12 (≈ 4-fold) activation in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Inhibition of MMP12 in JCR rats nearly completely blocked endostatin (≈ 85%) and angiostatin (≈ 90%) generation and significantly improved CCG (collateral-dependent zone flow was ≈ 66% of normal zone flow versus ≈ 12% for JCR RI). CONCLUSIONS: Compromised CCG in the metabolic syndrome is, in large part, because of increased MMP12 activation and consequent increased generation of endostatin and angiostatin, which inhibits late-stage collateral remodeling.