Endothelial Progenitor Cells and Macrophage Subsets Recruitment in Postischemic Mouse Hind Limbs.

INTRODUCTION: Peripheral arterial disease (PAD) occurs from atherosclerotic obstruction of arteries in the lower extremities. Restoration of perfusion requires angiogenesis and arteriogenesis through migration and differentiation of endothelial progenitor cells (EPCs) and macrophages at the site of injury. The time of recruitment has not been fully investigated. In this study, we investigated the infiltration of these cells in murine hind limb ischemia (HLI) model of PAD. METHODS: EPCs and M1-like and M2-like macrophages from ischemic skeletal muscles were quantified by flow cytometry at day-0, 1, 3, 7, and 14 post-HLI. RESULTS: The abundance of EPCs increased from day 1 and was highest on day 7 until day 14. M1-like population similarly increased and was highest on day 14 during the experiment. M2-like population was significantly greater than M1-like at baseline but surpassed the highest value of M1-like by day 7 during the experiment. Muscle regeneration and capillary density also increased and were highest at days 3 and 7, respectively, during the experiment. All mice achieved near full perfusion recovery by day 14. CONCLUSION: Thus, we observed a gradual increase in the percentage of EPC's and this was temporally paralleled with initial increase in M1-like followed by sustained increased in M2-like macrophages and perfusion recovered post-HLI.

BAG3 Attenuates Ischemia-Induced Skeletal Muscle Necroptosis in Diabetic Experimental Peripheral Artery Disease.

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.

Modulation of miR-29a and ADAM12 Reduces Post-Ischemic Skeletal Muscle Injury and Improves Perfusion Recovery and Skeletal Muscle Function in a Mouse Model of Type 2 Diabetes and Peripheral Artery Disease.

Both Type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2) are associated with an increased risk of limb amputation in peripheral arterial disease (PAD). How diabetes contributes to poor PAD outcomes is poorly understood but may occur through different mechanisms in DM1 and DM2. Previously, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. In an experimental PAD, we showed that ADAM12 is regulated by miR-29a and this regulation is impaired in ischemic endothelial cells in DM1, contributing to poor perfusion recovery. Here we investigated whether miR-29a regulation of ADAM12 is altered in experimental PAD in the setting of DM2. We also explored whether modulation of miR-29a and ADAM12 expression can improve perfusion recovery and limb function in mice with DM2. Our result showed that in the ischemic limb of mice with DM2, miR-29a expression is poorly downregulated and ADAM12 upregulation is impaired. Inhibition of miR-29a and overexpression of ADAM12 improved perfusion recovery, reduced skeletal muscle injury, improved muscle function, and increased cleaved Tie 2 and AKT phosphorylation. Thus, inhibition of miR-29a and or augmentation of ADAM12 improves experimental PAD outcomes in DM2 likely through modulation of Tie 2 and AKT signalling.

Dual specificity phosphatase 5 regulates perfusion recovery in experimental peripheral artery disease.

Peripheral artery disease (PAD) is caused by atherosclerotic occlusions of vessels outside the heart, particularly those of the lower extremities. Angiogenesis is one critical physiological response to vessel occlusion in PAD, but our understanding of the molecular mechanisms involved in angiogenesis is incomplete. Dual specificity phosphatase 5 (DUSP5) has been shown to play a key role in embryonic vascular development, but its role in post-ischemic angiogenesis is not known. We induced hind limb ischemia in mice and found robust upregulation of Dusp5 expression in ischemic hind limbs. Moreover, in vivo knockdown of Dusp5 resulted in impaired perfusion recovery in ischemic limbs and was associated with increased limb necrosis. In vitro studies showed upregulation of DUSP5 in human endothelial cells exposed to ischemia, and knockdown of DUSP5 in these ischemic endothelial cells resulted in impaired endothelial cell proliferation and angiogenesis, but did not alter apoptosis. Finally, we show that these effects of DUSP5 on post-ischemic angiogenesis are a result of DUSP5-dependent decrease in ERK1/2 phosphorylation and p21 protein expression. Thus, we have identified a role of DUSP5 in post-ischemic angiogenesis and implicated a DUSP5-ERK-p21 pathway that may serve as a therapeutic target for the modulation of post-ischemic angiogenesis in PAD.

Loss of interleukin-21 receptor activation in hypoxic endothelial cells impairs perfusion recovery after hindlimb ischemia.

OBJECTIVE: Surgical hindlimb ischemia (HLI) in mice has become a valuable preclinical model to study peripheral arterial disease. We previously identified that the different phenotypic outcomes after HLI across inbred mouse strains is related to a region on the short arm of mouse chromosome 7. The gene coding the interleukin-21 receptor (IL-21R) lies at the peak of association in this region. APPROACH AND RESULTS: With quantitative real-time polymerase chain reaction, we found that a mouse strain with a greater ability to upregulate IL-21R after HLI had better perfusion recovery than a strain with no upregulation after HLI. Immunofluorescent staining of ischemic hindlimb tissue showed IL-21R expression on endothelial cells (ECs) from C57BL/6 mice. An EC-enriched fraction isolated from ischemic hindlimb muscle showed higher Il-21R levels than an EC-enriched fraction from nonischemic limbs. In vitro, human umbilical vein ECs showed elevated IL-21R expression after hypoxia and serum starvation. Under these conditions, IL-21 treatment increased cell viability, decreased cell apoptosis, and augmented tube formation. In vivo, either knockout Il21r or blocking IL-21 signaling by treating with IL-21R-Fc (fusion protein that blocks IL-21 binding to its receptor) in C57BL/6 mice resulted in less perfusion recovery after HLI. Both in vitro and in vivo modulation of the IL-21/IL-21R axis under hypoxic conditions resulted in increased signal transducer and activator of transcription 3 phosphorylation and a subsequent increase in the B-cell lymphoma leukemia-2/BCL-2-associated X protein ratio. CONCLUSION: Our data indicate that IL-21R upregulation and ligand activation in hypoxic ECs may help perfusion recovery by limiting/preventing apoptosis and favoring cell survival and angiogenesis through the signal transducer and activator of transcription 3 pathway.

Endothelial interleukin-21 receptor up-regulation in peripheral artery disease.

In most patients with symptomatic peripheral artery disease (PAD), severe stenosis in or occlusion of the major blood vessels that supply the legs make the amount of distal blood flow dependent on the capacity to induce angiogenesis and collateral vessel formation. Currently, there are no medications that improve perfusion to the ischemic limb, and thus directly treat the primary problem of PAD. A recent report from our group in a pre-clinical mouse PAD model showed that interleukin-21 receptor (IL-21R) is up-regulated in the endothelial cells from ischemic hindlimb muscle. We further showed that loss of IL-21R resulted in impaired perfusion recovery in this model. In our study, we sought to determine whether IL-21R is present in the endothelium from ischemic muscle of patients with PAD. Using human gastrocnemius muscle biopsies, we found increased levels of IL-21R in the skeletal muscle endothelial cells of patients with PAD compared to control individuals. Interestingly, PAD patients had approximately 1.7-fold higher levels of circulating IL-21. These data provide direct evidence that the IL-21R pathway is indeed up-regulated in patients with PAD. This pathway may serve as a therapeutic target for modulation.

ADAM12: a genetic modifier of preclinical peripheral arterial disease.

In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.

MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway.

BACKGROUND: MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia. METHODS AND RESULTS: From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types. CONCLUSIONS: Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

Novel components in the nuclear factor-kappa B (NF-κB) signaling pathways of endothelial cells under hyperglycemic-ischemic conditions.

Diabetes worsens the outcomes of a number of vascular disorders including peripheral arterial disease (PAD) at least in part through induction of chronic inflammation. However, in experimental PAD, recovery requires the nuclear factor-kappa B (NF-κB) activation. Previously we showed that individually, both ischemia and high glucose activate the canonical and non-canonical arms of the NF-κB pathway, but prolonged high glucose exposure specifically impairs ischemia-induced activation of the canonical NF-κB pathway through activation of protein kinase C beta (PKCß). Although a cascade of phosphorylation events propels the NF-κB signaling, little is known about the impact of hyperglycemia on the canonical and non-canonical NF-κB pathway signaling. Moreover, signal upstream of PKCß that lead to its activation in endothelial cells during hyperglycemia exposure have not been well defined. In this study, we used endothelial cells exposed to hyperglycemia and ischemia (HGI) and an array of approximately 250 antibodies to approximately 100 proteins and their phosphorylated forms to identify the NF-κB signaling pathway that is altered in ischemic EC that has been exposed to high glucose condition. Comparison of signals from hyperglycemic and ischemic cell lysates yielded a number of proteins whose phosphorylation was either increased or decreased under HGI conditions. Pathway analyses using bioinformatics tools implicated BLNK/BTK known for B cell antigen receptor (BCR)-coupled signaling. Inhibition of BLNK/BTK in endothelial cells by a specific pharmacological inhibitor terreic acid attenuated PKC activation and restored the IκBα degradation suggesting that these molecules play a critical role in hyperglycemic attenuation of the canonical NF-κB pathway. Thus, we have identified a potentially new component of the NF-κB pathway upstream of PKC in endothelial cells that contributes to the poor post ischemic adaptation during hyperglycemia.

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia.

VEGF receptor (VEGFR) cell surface localization plays a critical role in transducing VEGF signaling toward angiogenic outcomes, and quantitative characterization of these parameters is critical to advancing computational models for predictive medicine. However, studies to this point have largely examined intact muscle; thus, essential data on the cellular localization of the receptors within the tissue are currently unknown. Therefore, our aims were to quantitatively analyze VEGFR localization on endothelial cells (ECs) from mouse hindlimb skeletal muscles after the induction of hindlimb ischemia, an established model for human peripheral artery disease. Flow cytometry was used to measure and compare the ex vivo surface localization of VEGFR1 and VEGFR2 on CD31(+)/CD34(+) ECs 3 and 10 days after unilateral ligation of the femoral artery. We determined that 3 days after hindlimb ischemia, VEGFR2 surface levels were decreased by 80% compared with ECs from the nonischemic limb; 10 days after ischemia, we observed a twofold increase in surface levels of the modulatory receptor, VEGFR1, along with increased proliferating cell nuclear antigen, urokinase plasminogen activator, and urokinase plasminogen activator receptor mRNA expression compared with the nonischemic limb. The significant upregulation of VEGFR1 surface levels indicates that VEGFR1 indeed plays a critical role in the ischemia-induced perfusion recovery process, a process that includes both angiogenesis and arteriogenesis. The quantification of these dissimilarities, for the first time ex vivo, provides insights into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors in ischemia and lays the foundation for systems biology approaches toward therapeutic angiogenesis.

Increased phosphodiesterase type 5 levels in a mouse model of type 2 diabetes mellitus.

INTRODUCTION: Diabetes mellitus (DM) is a major risk factor for developing erectile dysfunction (ED) and men with DM are often less responsive to phosphodiesterase type 5 (PDE5) inhibitors than ED due to other causes. AIMS: The aim of this study was to explore potential mechanisms whereby PDE5 inhibitors may have reduced efficacy in type 2 DM. METHODS: At 4 weeks of age, mice were either fed a high-fat diet (HFD) for 22-36 weeks or fed regular chow (control). An additional group of mice in the same genetic background had a genetic form of type 1 DM. MAIN OUTCOME MEASURES: Glucose tolerance testing, intracorporal pressures (ICPs), oxidative stress (OS), apoptotic cell death (active caspase-3 and apostain), PDE5, p53, and cyclic guanosine monophosphate (cGMP) levels, and histological examination of inflow arteries were performed in mice fed a HFD and control mice. A group of mice with type 1 DM were studied for PDE5 expression levels. RESULTS: All mice fed a HFD had impaired glucose tolerance compared with the age-matched mice fed on standard chow diet (control). HFD fed mice had reduced maximum ICPs following in vivo cavernous nerve electrical stimulation and increased apoptotic cell death, OS, and p53 levels in the corporal tissue. Interestingly, PDE5 levels were increased and cGMP levels were decreased. In contrast, mice with type 1 DM did not have increases in PDE5. CONCLUSIONS: Taken together, our results suggest that type 2 DM-induced ED is associated with findings that could lead to reduced cGMP and may account for reduced efficacy of PDE5 inhibitors.

Diabetes mellitus in peripheral artery disease: Beyond a risk factor.

Peripheral artery disease (PAD) is one of the major cardiovascular diseases that afflicts a large population worldwide. PAD results from occlusion of the peripheral arteries of the lower extremities. Although diabetes is a major risk factor for developing PAD, coexistence of PAD and diabetes poses significantly greater risk of developing critical limb threatening ischemia (CLTI) with poor prognosis for limb amputation and high mortality. Despite the prevalence of PAD, there are no effective therapeutic interventions as the molecular mechanism of how diabetes worsens PAD is not understood. With increasing cases of diabetes worldwide, the risk of complications in PAD have greatly increased. PAD and diabetes affect a complex web of multiple cellular, biochemical and molecular pathways. Therefore, it is important to understand the molecular components that can be targeted for therapeutic purposes. In this review, we describe some major developments in enhancing the understanding of the interactions of PAD and diabetes. We also provide results from our laboratory in this context.

Association between RAAS Antagonism and COVID-19-related Mortality in Patients with Overweight/Obesity-related Hypertension: A Retrospective Cohort Study. / Associação entre o Antagonismo do Sistema Renina-Angiotensina-Aldosterona e a Mortalidade Relacionada à COVID-19 em Pacientes com Hipertensão Relacionada ao Sobrepeso/Obesidade: um Estudo Retrospectivo de Coorte.

BACKGROUND: Angiotensin receptor blockers (ARB) and angiotensin-converting enzyme inhibitors (ACEI) increase the expression of ACE2, which is a receptor for entry of SARS-CoV-2 into cells. Though evidence suggests that ARB/ACEI are safe among the general population with COVID-19, their safety in patients with overweight/obesity-related hypertension deserves further evaluation. OBJECTIVE: We assessed the association between ARB/ACEI use and COVID-19 severity in patients with overweight/obesity-related hypertension. METHODS: This study included 439 adult patients with overweight/obesity (body mass index ≥ 25 kg/m2) and hypertension, diagnosed with COVID-19 and admitted to University of Iowa Hospitals and Clinic from March 1 to December 7, 2020. Mortality and severity of COVID-19 were evaluated based on length of stay in hospital, intensive care unit admission, use of supplemental oxygen, mechanical ventilation, and vasopressors. Multivariable logistic regression was used to examine the associations of ARB/ACEI use with mortality and other markers of COVID-19 severity, with a two-sided alpha set at 0.05. RESULTS: Exposure to ARB (n = 91) and ACEI (n = 149) before hospitalization was significantly associated with lower mortality (odds ratio [OR] = 0.362, 95% confidence interval [CI] 0.149 to 0.880, p = 0.025) and a shorter length of stay (95% CI -0.217 to -0.025, p = 0.015). Additionally, patients using ARB/ACEI showed a non-significant trend toward lower intensive care unit admission (OR = 0.727, 95% CI 0.485 to 1.090, p = 0.123), use of supplemental oxygen (OR = 0.929, 95% CI 0.608 to 1.421, p = 0.734), mechanical ventilation (OR = 0.728, 95% CI 0.457 to 1.161, p = 0.182), and vasopressors (OR = 0.677, 95% CI 0.430 to 1.067, p = 0.093). CONCLUSION: Results suggest that hospitalized patients with COVID-19 and overweight/obesity-related hypertension who were prescribed ARB/ACEI before admission to the hospital exhibit lower mortality and less severe COVID-19 than those who were not taking ARB/ACEI. The results also suggest that exposure to ARB/ACEI may protect patients with overweight/obesity-related hypertension from severe COVID-19 and death.

FUNDAMENTO: Os bloqueadores dos receptores da angiotensina (BRA) e os inibidores da enzima conversora da angiotensina (IECA) aumentam a expressão de ACE2, que é um receptor para entrada de SARS-CoV-2 nas células. Embora as evidências sugiram que os IECA/BRA são seguros entre a população geral com COVID-19, sua segurança em pacientes com hipertensão relacionada ao sobrepeso/obesidade merece uma avaliação mais aprofundada. OBJETIVO: Avaliamos a associação entre o uso de IECA/BRA e a gravidade da COVID-19 em pacientes com hipertensão relacionada ao sobrepeso/obesidade. MÉTODOS: O presente estudo incluiu 439 pacientes adultos com sobrepeso/obesidade (índice de massa corporal ≥ 25 kg/m2) e hipertensão, diagnosticados com COVID-19 e internados no University of Iowa Hospitals and Clinic entre 1º de março e 7 de dezembro de 2020. Foram avaliadas a mortalidade e a gravidade da COVID-19 com base no tempo de internação hospitalar, internação em unidade de terapia intensiva, uso de oxigênio suplementar, ventilação mecânica e uso de vasopressores. A regressão logística multivariável foi usada para examinar as associações do uso de IECA/BRA com a mortalidade e outros marcadores de gravidade de COVID-19, com um alfa bilateral definido em 0,05. RESULTADOS: A exposição aos BRA (n = 91) e IECA (n = 149) antes da hospitalização foi significativamente associada a menor mortalidade ( odds ratio [OR] = 0,362, intervalo de confiança [IC] de 95% 0,149 a 0,880, p = 0,025) e menor tempo de internação hospitalar (IC 95% −0,217 a −0,025, p = 0,015). Adicionalmente, os pacientes em uso de IECA/BRA apresentaram uma tendência não significativa de menor internação em unidade de terapia intensiva (OR = 0,727, IC 95% 0,485 a 1,090, p = 0,123), uso de oxigênio suplementar (OR = 0,929, IC 95% 0,608 a 1,421,p = 0,734), ventilação mecânica (OR = 0,728, IC 95% 0,457 a 1,161, p = 0,182) e vasopressores (OR = 0,677, IC 95% 0,430 a 1,067, p = 0,093). CONCLUSÃO: Os resultados sugerem que pacientes internados com COVID-19 e hipertensão relacionada ao sobrepeso/obesidade que receberam IECA/BRA antes da internação apresentam menor mortalidade e COVID-19 menos grave do que aqueles que não estavam tomando IECA/BRA. Os resultados também sugerem que a exposição aos IECA/BRA pode proteger pacientes com hipertensão relacionada ao sobrepeso/obesidade de COVID-19 grave e morte.

Alteration in angiogenic and anti-angiogenic forms of vascular endothelial growth factor-A in skeletal muscle of patients with intermittent claudication following exercise training.

The aims of this study were twofold: (1) to identify whether peripheral artery disease (PAD) patients had increased muscle concentration of angiogenic VEGF-A, anti-angiogenic VEGF165b or VEGF receptor 1 (VEGF-R1) when compared with control subjects, and (2) to evaluate whether exercise training in PAD patients was associated with changes in muscle concentration of VEGF-A, VEGF165b or VEGF-R1. At baseline, 22 PAD and 30 control subjects underwent gastrocnemius muscle biopsy. Twelve PAD patients were treated with supervised exercise training (SET) and underwent muscle biopsy after 3 weeks and 12 weeks of training and had sufficient tissue to measure VEGF-A, VEGF165b and VEGF-R1 concentrations in skeletal muscle lysates by ELISA. Muscle concentrations of VEGF-A and VEGF165b were similar in PAD patients versus controls at baseline. At both time points after the start of SET, VEGF-A levels decreased and there was a trend towards increased VEGF165b concentrations. At baseline, VEGF-R1 concentrations were lower in PAD patients when compared with controls but did not change after SET. Skeletal muscle concentrations of VEGF-A are not different in PAD patients when compared with controls at baseline. SET is associated with a significant reduction in VEGF-A levels and a trend towards increased VEGF165b levels. These somewhat unexpected findings suggest that further investigation into the mechanism of vascular responses to exercise training in PAD patients is warranted.

MicroRNA regulation of BAG3.

B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that modulates major biological processes, including apoptosis, autophagy, and development to promote cellular adaptive responses to stress stimuli. Although BAG3 is constitutively expressed in several cell types, its expression is also inducible and is regulated by microRNAs (miRNAs). miRNAs are small non-coding RNAs that mostly bind to the 3'-UTR (untranslated region) of mRNAs to inhibit their translation or to promote their degradation. miRNAs can potentially regulate over 50% of the protein-coding genes in a cell and therefore are involved in the regulation of all major functions, including cell differentiation, growth, proliferation, apoptosis, and autophagy. Dysregulation of miRNA expression is associated with pathogenesis of numerous diseases, including peripheral artery disease (PAD). BAG3 plays a critical role in regulating the response of skeletal muscle cells to ischemia by its ability to regulate autophagy. However, the biological role of miRNAs in the regulation of BAG3 in biological processes has only been elucidated recently. In this review, we discuss how miRNA may play a key role in regulating BAG3 expression under normal and pathological conditions.

microRNA-29a Regulates ADAM12 Through Direct Interaction With ADAM12 mRNA and Modulates Postischemic Perfusion Recovery.

Background Peripheral artery disease is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the postischemic adaptation involved in restoring blood flow in peripheral artery disease. Previously, in a murine hind limb ischemia model of peripheral artery disease, we identified ADAM12 (a disintegrin and metalloproteinase gene 12) as a key genetic modifier of postischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MicroRNAs are a class of small, noncoding, single-stranded RNAs that regulate gene expression primarily through transcriptional repression of messenger RNA (mRNA). We showed microRNA-29a (miR-29a) modulates ADAM12 expression in the setting of diabetes and ischemia. However, how miR-29a modulates ADAM12 is not known. Moreover, the physiological effects of miR-29a modulation in a nondiabetic setting is not known. Methods and Results We overexpressed or inhibited miR-29a in ischemic mouse gastrocnemius and tibialis anterior muscles, and quantified the effect on perfusion recovery, ADAM12 expression, angiogenesis, and skeletal muscle regeneration. In addition, using RNA immunoprecipitation-based anti-miR competitive assay, we investigated the interaction of miR-29a and ADAM12 mRNA in mouse microvascular endothelial cell, skeletal muscle, and human endothelial cell lysates. Ectopic expression of miR-29a in ischemic mouse hind limbs decreased ADAM12 mRNA expression, increased skeletal muscle injury, decreased skeletal muscle function, and decreased angiogenesis and perfusion recovery, with no effect on skeletal muscle regeneration and myofiber cross-sectional area following hind limb ischemia. RNA immunoprecipitation-based anti-miR competitive assay studies showed miR-29a antagomir displaced miR-29a and ADAM12 mRNA from the AGO-2 (Argonaut-2) complex in a dose dependent manner. Conclusions Taken together, the data show miR-29a suppresses ADAM12 expression by directly binding to its mRNA, resulting in impaired skeletal muscle function, angiogenesis, and poor perfusion. Hence, elevated levels of miR-29a, as seen in diabetes and aging, likely contribute to vascular pathology, and modulation of miR-29a could be a therapeutic target.

Peripheral arterial disease in diabetes: is there a role for genetics?

Atherosclerotic occlusion of vessels outside of the heart is commonly referred to as peripheral arterial disease (PAD). The lower extremity is the most common site of PAD and its development is associated with the same risk factors involved in general atherosclerosis. However, there is emerging evidence that other risk factors may play a key role in the development of PAD. Over the past decade polymorphism in a number of genes has been shown to contribute to the risk of developing PAD. These genes can be classified into proartherosclerosis or proatherothrombosis based on the known gene function. Moreover, they can be categorized as "novel" polymorphism when the function of the genes is not known or when the specific gene within an associated genetic locus is not known. It is intriguing that not only are gene polymorphisms associated with PAD being identified, but more recently studies are now finding gene polymorphisms that may be important in development of this syndrome only in the contest of certain environmental factors such as diabetes. Currently how these gene-environment interactions contribute to the pathogenesis of PAD is poorly understood but will likely play a critical role in future understanding of this complex disease.

Lessons learned from glycemia control studies.

Hyperglycemia occurs in patients with diabetes and in nondiabetic patients during acute illness. Epidemiologic and observational studies have demonstrated that hyperglycemia is associated with significant adverse outcomes. Nevertheless, studies evaluating the benefits of normalizing glycemia have produced inconsistent results. For instance, intensive control of hyperglycemia had been shown to provide microvascular benefit in type 1 and type 2 diabetic patients, but its macrovascular benefits had only been clearly demonstrated in type 1 diabetic patients. Moreover, although initial studies in critically ill patients showed decreased morbidity and mortality with tight glycemic control, subsequent studies yielded conflicting results. A series of recent studies provide further insight and show that intensive glycemic control in type 2 diabetic patients does provide macrovascular benefit but is associated with increased risk of hypoglycemia. In the critically ill patient, tight glycemic control could be detrimental; thus, a less aggressive glycemic target of 140 to 180 mg/dL is preferred.

Inhibition of protein kinase C beta phosphorylation activates nuclear factor-kappa B and improves postischemic recovery in type 1 diabetes.

IMPACT STATEMENT: Diabetes worsens the outcomes of peripheral arterial disease (PAD) likely in part through inducing chronic inflammation. However, in PAD, recovery requires the nuclear factor-kappa B (NF-κB) activation, a known contributor to inflammation. Our study shows that individually, both ischemia and high glucose activate the canonical and non-canonical arms of the NF-κB pathways. We show for the first time that prolonged high glucose specifically impairs ischemia-induced activation of the canonical NF-κB pathway through activation of protein kinase C beta (PKCß). Accordingly, inhibition of PKCß restores the ischemia-induced NF-κB activity both in vitroin endothelial cells and in vivoin hind limbs of type 1 diabetic mice and improves perfusion recovery after experimental PAD. Thus, this study provides a mechanistic insight into how diabetes contributes to poor outcomes in PAD and a potential translational approach to improve PAD outcomes.

Individuals with Peripheral Artery Disease (PAD) and Type 1 Diabetes Are More Likely to Undergo Limb Amputation than Those with PAD and Type 2 Diabetes.

BACKGROUND: Limited data exist comparing how type 1 diabetes mellitus (DM) and type 2 DM may have differential effects on peripheral artery disease (PAD) severity. We aimed to study the association of type of DM with the procedure utilized in hospitalizations with a diagnosis of PAD. METHODS: We used the national inpatient sample databases from 2003 to 2014 to identify hospitalizations with a diagnosis of PAD and type 1 or type 2 DM. Logistic regression was utilized to evaluate the association between type of DM and procedure utilized (amputation-overall, major, endovascular revascularization, surgical revascularization). RESULTS: We identified 14,012,860 hospitalizations with PAD diagnosis and DM, 5.6% (n = 784,720) had type 1 DM. The patients with type 1 DM were more likely to present with chronic limb-threatening ischemia (CLTI) (45.2% vs. 32.0%), ulcer (25.9% vs. 17.7%), or complicated ulcer (16.6% vs. 10.5%) (all p < 0.001) when compared to those with type 2 DM. Type 1 DM was independently and significantly associated with more amputation procedures (adjusted odds ratio = 1.12, 95% confidence interval [CI] I 1.08 to 1.16, p < 0.001). Overall, in-hospital mortality did not differ between the individuals with type 1 and type 2 DM. The overall mean (95% CI) length of stay (in days) was 6.6 (6.5 to 6.6) and was significantly higher for type 1 DM (7.8 [7.7 to 8.0]) when compared to those with type 2 DM (6.5 [6.4 to 6.6]). CONCLUSION: We observed that individuals with PAD and type 1 DM were more likely to present with CLTI and ulcer and undergo amputation when compared to those with PAD and type 2 diabetes. Further studies are needed to better understand the underlying mechanisms behind these findings and to identify novel interventions to reduce the risk of amputation in patients with type 1 DM.