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 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.

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.

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 / Association between RAAS Antagonism and COVID-19-related Mortality in Patients with Overweight/Obesity-related Hypertension: A Retrospective Cohort Study

Resumo 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.

Abstract 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.

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.

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.

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.

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.

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.

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.

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.

In experimental peripheral arterial disease, type 2 diabetes alters post-ischemic gene expression.

Peripheral arterial disease is characterized by impaired blood flow to tissues outside the heart due to atherosclerosis and it most frequently occurs in the lower extremities. Type 2 diabetes (T2D) is a well-known risk factor that accelerate the course and contributes to poor clinical outcomes of PAD. While there is some evidence that T2D is associated with altered expression of genes involved in regulating PAD severity, our knowledge about the specific genes and pathways involved remains incomplete. We induced experimental PAD or hind limb ischemia in T2D and non-diabetic mice and subjected the ischemic gastrocnemius muscle tissues to genome-wide mRNA transcriptome analysis. We subsequently performed pathway analysis on the top 500 genes that showed the most significant expression differences between the ischemic diabetic and ischemic non-diabetic muscle tissues. Pathway analysis of the differentially expressed genes identified pathways involved in essential biological processes such as "metabolic pathways," "phagosomes," "lysosomes," and "regulation of actin cytoskeleton". Overall, our data provides the opportunity to test hypotheses on the potential role of the altered genes/molecular pathways in poor PAD outcomes in diabetes.

Type 1 diabetes alters ischemia-induced gene expression.

Peripheral Artery Disease (PAD) is a chronic, activity-limiting disease that is caused by atherosclerotic occlusion of blood vessels outside the heart. Type 1 Diabetes (T1D) not only increases an individual's likelihood of developing PAD, but also contributes to poor clinical outcomes after PAD manifestation. Although there is some evidence suggesting that hyperglycemia might alter expression of genes involved in regulating PAD severity or outcomes, our knowledge about the specific genes and pathways involved remains incomplete. We induced experimental PAD or hind limb ischemia in T1D and non-diabetic mice and subjected the ischemic gastrocnemius muscle tissues to genome-wide mRNA transcriptome and pathway analysis. We identified 513 probe sets that represented 443 different genes with highly significant expression differences (p < 0.005) between the ischemic diabetic and ischemic non-diabetic muscle tissues. Moreover, pathway analysis of the differentially expressed genes identified pathways involved in essential biological processes such as "cell cycle," "DNA replication," "metabolic pathways," "focal adhesion," "regulation of actin cytoskeleton," and "nucleotide excision repair". Taken together, our data offer the opportunity to test hypotheses on the roles played by the altered genes/molecular pathways in poor PAD outcomes in diabetes. Such studies may lead to the development of specific therapies to improve PAD outcomes in patients with comorbid diabetes.

Role of genetics in peripheral arterial disease outcomes; significance of limb-salvage quantitative locus-1 genes.

Peripheral artery disease is a major health care problem with significant morbidity and mortality. Humans with peripheral artery disease exhibit two major and differential clinical manifestations - intermittent claudication and critical limb ischemia. Individuals with intermittent claudication or critical limb ischemia have overlapping risk factors and objective measures of blood flow. Hence, we hypothesized that variation in genetic make-up may be an important determinant in the severity of peripheral artery disease. Previous studies have identified polymorphism in genes, contributing to extent of atherosclerosis but much less is known about polymorphisms associated with genes that can influence peripheral artery disease severity. This review outlines some of the progress made up-to-date to unravel the molecular mechanisms underlining differential peripheral artery disease severity. By exploring the recovery phenotype of different mouse strains following experimental peripheral artery disease, our group identified the limb salvage-associated quantitative trait locus 1 on mouse chromosome 7 as the first genetic modifier of perfusion recovery and tissue necrosis phenotypes. Furthermore, a number of genes within LSq-1, such as ADAM12, IL-21Rα, and BAG3 were identified as genetic modifiers of peripheral artery disease severity that function through preservation of endothelial and skeletal muscle cells during ischemia. Taken together, these studies suggest manipulation of limb salvage-associated quantitative trait locus 1 genes show great promise as therapeutic targets in the management of peripheral artery disease. Impact statement Peripheral artery disease (PAD) is a major health care problem with significant morbidity and mortality. Individuals with similar atherosclerosis burden do display different severity of disease. This review outlines some of the progress made up-to-date in unraveling the molecular mechanisms underlining differential PAD severity with a focus on the role of the Limb Salvage-associated Quantitative trait locus 1 (LSq-1), a key locus in adaptation to ischemia in PAD.

Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia.

Individuals with diabetes mellitus suffer from impaired angiogenesis and this contributes to poorer peripheral arterial disease outcomes. In experimental peripheral arterial disease, angiogenesis and perfusion recovery are impaired in mice with diabetes. We recently showed that a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12) is upregulated in ischemic endothelial cells and plays a key role in post-ischemic angiogenesis and perfusion recovery following experimental peripheral arterial disease. Here we investigated the role of miR29a in the regulation of endothelial cell ADAM12 expression in ischemia and how hyperglycemia negatively affects this regulation. We also explored whether modulating miR29a can improve impaired post-ischemic angiogenesis associated with hyperglycemia. Additionally, we tested whether miR29a modulation could improve post ischemic angiogenesis in the setting of impaired vascular endothelial growth factor signaling. We forced miR29a expression in ischemic endothelial cells and assessed ADAM12 expression. We also evaluated whether hyperglycemia in vivo and in vitro impair ischemia-induced ADAM12 upregulation and miR29a downregulation. Lastly, we determined whether modulating endothelial cell miR29a expression in ischemia and hyperglycemia could improve impaired endothelial cell functions. We found under ischemic conditions where ADAM12 is upregulated in endothelial cells, miR29a is downregulated. Forced expression of miR29a in ischemic endothelial cell prevented ADAM12 upregulation . In ischemic hind limbs of mice with type 1 diabetes and in endothelial cells exposed to simulated ischemia plus hyperglycemia, ADAM12 upregulation and miR29a downregulation were blunted while angiogenesis was impaired. Knocking down miR29a with an miR29a inhibitor was sufficient to improve ADAM12 upregulation and angiogenesis in simulated ischemia plus hyperglycemia. It was also sufficient to improve perfusion recovery in type 1 diabetes mellitus mice in vivo and angiogenesis in vitro even when vascular endothelial growth factor signaling was impaired with blocking antibodies. In conclusion, MiR29a regulates endothelial cell ADAM12 upregulation in ischemia and this is impaired in hyperglycemia. Modulating miR29a improves impaired post-ischemic angiogenesis associated with hyperglycemia. Impact statement Individuals with diabetes are more likely to develop peripheral arterial disease (PAD), and when PAD is present, in those with diabetes, it is more severe and there is currently no effective medical treatment for impaired blood flow which occurs in diabetics with PAD. The current work advances the field by providing an understanding of a molecular mechanism involved in impaired post ischemic angiogenesis in diabetes. It shows for the first time that failure to downregulate miR29a in ischemic diabetic tissues is a major contributing factor to poor perfusion recovery in experimental PAD, and miR29a is elevated in skeletal muscle samples from human diabetics compared with levels in those without diabetes. Knocking down the elevated miR29a in ischemic diabetic mouse hind limbs improved perfusion recovery following experimental PAD. This shows miR29a modulation as a novel therapeutic target for improving blood flow in diabetics with PAD.

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.

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.

Glycaemic control improves perfusion recovery and VEGFR2 protein expression in diabetic mice following experimental PAD.

AIMS: Diabetes mellitus (DM) is associated with poor clinical outcomes in humans with peripheral arterial disease (PAD) and in pre-clinical models of PAD, but the effects of glycaemic control are poorly understood. We investigated the effect of glycaemic control on experimental PAD in mice with Type 1 DM and explored the effects of hyperglycaemia on vascular endothelial growth factor receptor 2 (VEGFR2) expression in ischaemia. METHODS AND RESULTS: Hind limb ischaemia was induced in non-diabetic, untreated Type 1 DM, and treated Type 1 DM mice. We assessed perfusion recovery, capillary density, VEGFR2 levels, and VEGFR2 ubiquitination in ischaemic hind limbs. We found that untreated Type 1 DM mice showed impaired perfusion recovery, lower hind limb capillary density 5 weeks post-ischaemia, and lower VEGFR2 protein in Day 3 post-ischaemic hind limbs when compared with non-DM controls. Treated Type 1 DM mice had perfusion recovery, capillary density, and VEGFR2 protein levels comparable with that of non-diabetic mice at the same time points. Treatment with anti-VEGFR2 antibody negated that the improved perfusion recovery displayed by treated Type 1 DM mice. In ischaemic Type 1 DM hind limbs and endothelial cells exposed to simulated ischaemia, high glucose impaired VEGFR2 expression and was associated with increased VEGFR2 ubiquitination. Inhibition of the ubiquitin-proteasome complex restored normal endothelial VEGFR2 expression in simulated ischaemia. CONCLUSION: Hyperglycaemia in Type 1 DM impairs VEGFR2 protein expression in ischaemic hind limbs, likely due to increased ubiquitination and degradation by the proteasome complex. Glycaemic control allows normal levels of VEGFR2 in ischaemia and improved perfusion recovery.