COVID-19 Associated Cardiovascular Disease-Risks, Prevention and Management: Heart at Risk Due to COVID-19.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) virus and the resulting COVID-19 pandemic have had devastating and lasting impact on the global population. Although the main target of the disease is the respiratory tract, clinical outcomes, and research have also shown significant effects of infection on other organ systems. Of interest in this review is the effect of the virus on the cardiovascular system. Complications, including hyperinflammatory syndrome, myocarditis, and cardiac failure, have been documented in the context of COVID-19 infection. These complications ultimately contribute to worse patient outcomes, especially in patients with pre-existing conditions such as hypertension, diabetes, or cardiovascular disease (CVD). Importantly and interestingly, reports have demonstrated that COVID-19 also causes myocardial injury in adults without pre-existing conditions and contributes to systemic complications in pediatric populations, such as the development of multisystem inflammatory syndrome in children (MIS-C). Although there is still a debate over the exact mechanisms by which such complications arise, understanding the potential paths by which the virus can influence the cardiovascular system to create an inflammatory environment may clarify how SARS-CoV-2 interacts with human physiology. In addition to describing the mechanisms of disease propagation and patient presentation, this review discusses the diagnostic findings and treatment strategies and the evolution of management for patients presenting with cardiovascular complications, focusing on disease treatment and prevention.

Biodegradable piezoelectric skin-wound scaffold.

Electrical stimulation (ES) induces wound healing and skin regeneration. Combining ES with the tissue-engineering approach, which relies on biomaterials to construct a replacement tissue graft, could offer a self-stimulated scaffold to heal skin-wounds without using potentially toxic growth factors and exogenous cells. Unfortunately, current ES technologies are either ineffective (external stimulations) or unsafe (implanted electrical devices using toxic batteries). Hence, we propose a novel wound-healing strategy that integrates ES with tissue engineering techniques by utilizing a biodegradable self-charged piezoelectric PLLA (Poly (l-lactic acid)) nanofiber matrix. This unique, safe, and stable piezoelectric scaffold can be activated by an external ultrasound (US) to produce well-controlled surface-charges with different polarities, thus serving multiple functions to suppress bacterial growth (negative surface charge) and promote skin regeneration (positive surface charge) at the same time. We demonstrate that the scaffold activated by low intensity/low frequency US can facilitate the proliferation of fibroblast/epithelial cells, enhance expression of genes (collagen I, III, and fibronectin) typical for the wound healing process, and suppress the growth of S. aureus and P. aeruginosa bacteria in vitro simultaneously. This approach induces rapid skin regeneration in a critical-sized skin wound mouse model in vivo. The piezoelectric PLLA skin scaffold thus assumes the role of a multi-tasking, biodegradable, battery-free electrical stimulator which is important for skin-wound healing and bacterial infection prevention simultaneuosly.

Role of Pellino-1 in Inflammation and Cardioprotection following Severe Sepsis: A Novel Mechanism in a Murine Severe Sepsis Model †.

OBJECTIVES: Intra-abdominal sepsis is commonly diagnosed in the surgical population and remains the second most common cause of sepsis overall. Sepsis-related mortality remains a significant burden in the intensive care unit despite advances in critical care. Nearly a quarter of the deaths in people with heart failure are caused by sepsis. We have observed that overexpression of mammalian Pellino-1 (Peli1), an E3 ubiquitin ligase, causes inhibition of apoptosis, oxidative stress, and preservation of cardiac function in a myocardial infarction model. Given these manifold applications, we investigated the role of Peli1 in sepsis using transgenic and knockout mouse models specific to this protein. Therefore, we aimed to explore further the myocardial dysfunction seen in sepsis through its relation to the Peli 1 protein by using the loss of function and gain-of-function strategy. METHODS: A series of genetic animals were created to understand the role of Peli1 in sepsis and the preservation of heart function. Wild-type, global Peli1 knock out (Peli1-/-), cardiomyocyte-specific Peli1 deletion (CP1KO), and cardiomyocyte-specific Peli1 overexpressing (alpha MHC (αMHC) Peli1; AMPEL1Tg/+) animals were divided into sham and cecal ligation and puncture (CLP) surgical procedure groups. Cardiac function was determined by two-dimensional echocardiography pre-surgery and at 6- and 24-h post-surgery. Serum IL-6 and TNF-alpha levels (ELISA) (6 h), cardiac apoptosis (TUNEL assay), and Bax expression (24 h) post-surgery were measured. Results are expressed as mean ± S.E.M. RESULTS: AMPEL1Tg/+ prevents sepsis-induced cardiac dysfunction assessed by echocardiographic analysis, whereas global and cardiomyocyte-specific deletion of Peli1 shows significant deterioration of cardiac functions. Cardiac function was similar across the sham groups in all three genetically modified mice. ELISA assay displayed how Peli 1 overexpression decreased cardo-suppressive circulating inflammatory cytokines (TNF-alpha, IL-6) compared to both the knockout groups. The proportion of TUNEL-positive cells varied according to Peli1 expression, with overexpression (AMPEL1Tg/+) leading to a significant reduction and Peli1 gene knockout (Peli1-/- and CP1KO) leading to a significant increase in their presence. A similar trend was also observed with Bax protein expression. The improved cellular survival associated with Peli1 overexpression was again shown with the reduction of oxidative stress marker 4-Hydroxy-2-Nonenal (4-HNE). CONCLUSION: Our results indicate that overexpression of Peli1 is a novel approach that not only preserved cardiac function but reduced inflammatory markers and apoptosis following severe sepsis in a murine genetic model.

Driving adult tissue repair via re-engagement of a pathway required for fetal healing.

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and ß-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

Protective Effect of Cardiomyocyte-Specific Prolyl-4-Hydroxylase 2 Inhibition on Ischemic Injury in a Mouse MI Model.

BACKGROUND: Our earlier studies showed that inhibiting prolyl-4-hydroxylase enzymes (PHD-1 and PHD-3) improves angiogenesis, heart function, and limb perfusion in mouse models via stabilizing hypoxia-inducible transcription factor-alpha (HIF-1α). The present study explored the effects of the prolyl-4-hydroxylase enzyme, PHD-2, on ischemic heart failure using cardiac-specific PHD-2 gene knockout (KO) mice (PHD2 -/- ). STUDY DESIGN: Adult wild-type (WT) and PHD2 -/- mice, 8-12 weeks old, were subjected to myocardial infarction (MI) by irreversibly ligating the left anterior descending (LAD) coronary artery. All sham group mice underwent surgery without LAD ligation. Animals were divided into 4 groups: (1) wild-type sham (WTS); (2) wild-type myocardial infarction (WTMI); (3) PHD2KO sham (PHD2 -/- S); (4) PHD2KO myocardial infarction (PHD2 -/- MI). Left ventricular tissue samples collected at various time points after surgery were used for microRNA expression profiling, Western blotting, and immunohistochemical analysis. RESULTS: Volcano plot analysis revealed 19 differentially-expressed miRNAs in the PHD2 -/- MI group compared with the WTMI group. Target analysis using Ingenuity Pathway Analysis showed several differentially regulated miRNAs targeting key signaling pathways such as Akt, VEGF, Ang-1, PTEN, apoptosis, and hypoxia pathways. Western blot analysis showed increased HIF-1α, VEGF, phospho-AKT, ß-catenin expression and reduced Bax expression for the PHD2 -/- MI group compared with the WTMI group. Echocardiographic analysis showed preserved heart functions, and picrosirius red staining revealed decreased fibrosis in PHD2 -/- MI compared with the WTMI group. CONCLUSIONS: PHD2 inhibition showed preserved heart function, enhanced angiogenic factor expression, and decreased apoptotic markers after MI. Overall, cardiac PHD2 gene inhibition is a promising candidate for managing cardiovascular diseases.

Gene therapy with Pellino-1 improves perfusion and decreases tissue loss in Flk-1 heterozygous mice but fails in MAPKAP Kinase-2 knockout murine hind limb ischemia model.

OBJECTIVES: In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/--) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/- and, MAPKAPKINASE2 (MK2-/-) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. STUDY DESIGN AND METHODS: Femoral artery ligation (HLI) was performed in both Flk-1+/- and MK2-/- mice along with their corresponding WT. Another set of Flk-1+/- and MK2-/- were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. RESULTS: Flk-1+/- and MK2-/- had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/- and MK2-/- KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/- limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2-/- ischemic limb after Ad. Peli1 injection. CONCLUSION: Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/- mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD.

Heat shock protein A12B gene therapy improves perfusion, promotes neovascularization, and decreases fibrosis in a murine model of hind limb ischemia.

BACKGROUND: Heat shock protein A12B expressed in endothelial cells is important and required for angiogenesis to form functional vessels in ischemic tissue. We have previously shown the cardioprotective effects of heat shock protein A12B overexpression in a rat model of diabetic myocardial infarction. In this study, we aim to explore the role of heat shock protein A12B in a surgically-induced murine hind-limb ischemia model. MATERIALS AND METHODS: Adult 8- to 12-week-old C57BL/6J mice were divided into 2 groups: treated with Adeno.LacZ (control group) and with Adeno.HSPA12B (experimental group) and, with both groups subjected to right femoral artery ligation. Immediately after surgery, mice in both groups received either Adeno.HSPA12B or Adeno.LacZ (1 × 109 plaque forming units) in both the semimembranosus and gastrocnemius muscles of the right limb. The left limb served as the internal control. Both groups underwent serial laser Doppler imaging preoperatively, and again postoperatively until 28 days. Immunohistochemical analysis was performed 3 and 28 days post-surgery. RESULTS: Mice in the Adeno.HSPA12B gene therapy group showed improved motor function and a significantly higher blood perfusion ratio on postoperative days 21 and 28, along with better motor function. Immunohistochemical analysis showed increased expression of vascular endothelial growth factor, thioredoxin-1, heme oxygenase, and hypoxia-inducible factor 1α, along with a decreased expression of A-kinase-anchoring protein 12 and thioredoxin-interacting protein levels. The Adeno.HSPA12B-treated group also showed increased capillary and arteriolar density and an increased capillary-myocyte ratio, along with reduced fibrosis compared to the Adeno.LacZ group. CONCLUSION: Our study demonstrates that targeted Adeno.HSPA12B gene delivery into ischemic muscle enhances perfusion and angiogenic protein expression. This molecule shows promise for the management of peripheral vascular disease as a potential target for clinical trials and subsequent drug therapy.

Engineered resveratrol-loaded fibrous scaffolds promotes functional cardiac repair and regeneration through Thioredoxin-1 mediated VEGF pathway.

Despite recent advancements, mortality due to coronary heart disease (CHD) remains high. Recently, the use of tissue-engineered grafts and scaffolds has emerged as a candidate for supporting the myocardium after an ischemic event. Resveratrol is a naturally occurring plant-based non-flavonoid polyphenolic compound found in many natural foods, including grapes and red wine. We embedded resveratrol in a polycaprolactone (PCL) scaffold and evaluated the cardio-therapeutic effects in a murine model of myocardial infarction (MI), with animals being grouped into Sham (S), Myocardial Infarction (MI), MI + PCL, and MI + PCL-Resveratrol (MI + PCL-R). After 4 and 8 weeks, echocardiography was performed to assess ejection fraction (EF) and fractional shortening (FS), which was followed by immunohistochemistry and immunofluorescence analysis at 8 weeks. The MI + PCL-R group showed a significant improvement in EF and FS compared with the MI + PCL group at 4 and 8-weeks post-surgery. PCL-R scaffolds treated hearts revealed decreased inflammatory cell infiltration, improved collagen extracellular matrix (ECM) secretion and blood vessel network formation following MI. The immunofluorescence analysis revealed resveratrol-loaded scaffolds promote increased expression of cTnT, Cx-43, Trx-1, and VEGF proteins. This study reports resveratrol-mediated rescue of ischemic myocardium when delivered through a biodegradable polymeric scaffold system after MI.

Deletion of newly described pro-survival molecule Pellino-1 increases oxidative stress, downregulates cIAP2/NF-κB cell survival pathway, reduces angiogenic response, and thereby aggravates tissue function in mouse ischemic models.

INTRODUCTION: In the present study, we aimed to explore the functional role of Pellino-1 (Peli1) in inducing neovascularization after myocardial infarction (MI) and hindlimb ischemia (HLI) using Peli1 global knockout mice (Peli1-/-). Recently we have shown that Peli1, an E3 ubiquitin ligase, induce angiogenesis and improve survivability, with decreased necrosis of ischemic skin flaps. METHODS: Peli1fl/fl and Peli1-/- mice were subjected to either permanent ligation of the left anterior descending coronary artery (LAD) or sham surgery (S). Tissues from the left ventricular risk area were collected at different time points post-MI. In addition, Peli1fl/fl and Peli1-/- mice were also subjected to permanent ligation of the right femoral artery followed by motor function scores, Doppler analysis for blood perfusion and immunohistochemical analysis. RESULTS: Global Peli1 knockout exacerbated myocardial dysfunction, 30 and 60 days after MI compared to wild type (WT) mice as measured by echocardiogram. In addition, Peli1-/- mice also showed decreased motor function scores and perfusion ratios compared with Peli1fl/fl mice 28 days after the induction of HLI. The use of Peli1 in adenoviral gene therapy following HLI in CD1 mice improved the perfusion ratio at 28 days compared to Ad.LacZ-injected mice. CONCLUSION: These results suggest new insights into the protective role of Peli1 on ischemic tissues and its influence on survival signaling.

Evaluation of dermal tissue regeneration using resveratrol loaded fibrous matrix in a preclinical mouse model of full-thickness ischemic wound.

The complete loss of dermal tissue due to ischemia is a serious challenge facing clinicians. Frequently, the failure of wound healing is due to ischemic conditions prevailing at the site of damaged tissue. Restoration of lost vasculature at the ischemic site can be achieved by supplementing proangiogenic stimuli through an engineered scaffold mimicking dermal extracellular matrix. Towards this objective, we have developed an electrospun scaffold loaded with the pro-angiogenic molecule resveratrol. The physical and chemical changes in the polymeric scaffold before and after loading of resveratrol were characterized using field emission scanning electron microscopy (FE-SEM), Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), coherence scanning interferometry (CSI) and X-ray diffraction (XRD). A sustained release of resveratrol from the scaffold was elucidated by UV-spectrophotometer analysis. The enhancement in cell-matrix interaction was studied using human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds. The biocompatibility analysis of resveratrol loaded scaffolds was evaluated through a subcutaneous implantation study in mice. The therapeutic potential of resveratrol loaded scaffolds to accelerate tissue repair was analyzed in a full-thickness ischemic wound model in mice. Wound closure and H&E staining analysis showed rapid closure of ischemic wound area and re-epithelialization in resveratrol loaded scaffold treated groups compared to collagen and negative control groups. The immunostaining analysis further revealed the activation of thioredoxin-1 (Trx-1), heme oxygenase-1 (HO-1) mediated vascular endothelial growth factor (VEGF) signaling in resveratrol loaded scaffold treated group. The expression of Bcl-2 in healing wound edges post-treatment with resveratrol loaded scaffold confirmed the anti-apoptotic effect mediated by resveratrol. From this study, we explored a synergistic effect mediated by resveratrol and fibrous scaffolds to aid the ischemic wound healing process through effective vascularization.

Disruption of VEGF Mediated Flk-1 Signaling Leads to a Gradual Loss of Vessel Health and Cardiac Function During Myocardial Infarction: Potential Therapy With Pellino-1.

Background The present study demonstrates that the ubiquitin E3 ligase, Pellino-1 (Peli1), is an important angiogenic molecule under the control of vascular endothelial growth factor (VEGF) receptor 2/Flk-1. We have previously reported increased survivability of ischemic skin flap tissue by adenovirus carrying Peli1 (Ad-Peli1) gene therapy in Flk-1+/- mice. Methods and Results Two separate experimental groups of mice were subjected to myocardial infarction ( MI ) followed by the immediate intramyocardial injection of adenovirus carrying LacZ (Ad-LacZ) (1×109 pfu) or Ad-Peli1 (1×109 pfu). Heart tissues were collected for analyses. Compared with wild-type ( WTMI ) mice, analysis revealed decreased expressions of Peli1, phosphorylated (p-)Flk-1, p-Akt, p- eNOS , p- MK 2, p-IκBα, and NF -κB and decreased vessel densities in Flk-1+/- mice subjected to MI (Flk-1+/- MI ). Mice ( CD 1) treated with Ad-Peli1 after the induction of MI showed increased ß-catenin translocation to the nucleus, connexin 43 expression, and phosphorylation of Akt, eNOS , MK 2, and IκBα, that was followed by increased vessel densities compared with the Ad-LacZ-treated group. Echocardiography conducted 30 days after surgery showed decreased function in the Flk1+/- MI group compared with WTMI , which was restored by Ad-Peli1 gene therapy. In addition, therapy with Ad-Peli1 stimulated angiogenic and arteriogenic responses in both CD 1 and Flk-1+/- mice following MI . Ad-Peli1 treatment attenuated cardiac fibrosis in Flk-1+/- MI mice. Similar positive results were observed in CD 1 mice subjected to MI after Ad-Peli1 therapy. Conclusion Our results show for the first time that Peli1 plays a unique role in salvaging impaired collateral blood vessel formation, diminishes fibrosis, and improves myocardial function, thereby offering clinical potential for therapies in humans to mend a damaged heart following MI .

Thioredoxin-1 augments wound healing and promote angiogenesis in a murine ischemic full-thickness wound model.

BACKGROUND: Nonhealing wounds are a continuing health problem in the United States. Overproduction of reactive oxygen species is a major causative factor behind delayed wound healing. Previously we reported that thioredoxin-1 treatment could alleviate oxidative stress under ischemic conditions, such as myocardial infarction and hindlimb ischemia. In this study, we explored the potential for thioredoxin-1 gene therapy to effectively aid wound healing through improved angiogenesis in a murine ischemic wound model. METHODS: Full-thickness, cutaneous, ischemic wounds were created in the dorsum skin flap of 8- to 12-week-old CD1 mice. Nonischemic wounds created lateral to the ischemic skin flap served as internal controls. Mice with both ischemic wounds and nonischemic wounds were treated with Adeno-LacZ (1 × 109 pfu) or Adeno-thioredoxin-1 (1 × 109 pfu), injected intradermally around the wound. Digital imaging was performed on days 0, 3, 6, and 9 to assess the rate of wound closure. Tissue samples collected at predetermined time intervals were processed for immunohistochemical analysis. RESULTS: No significant differences in wound closure were identified among the nonischemic wounds control, nonischemic wounds-LacZ, and nonischemic wounds-thioredoxin-1 groups. Hence, only mice with ischemic wounds were further analyzed. The ischemic wounds-thioredoxin-1 group had significant improvement in wound closure on days 6 and 9 after surgery compared with the ischemic wounds control and ischemic wounds-LacZ groups. Immunohistochemical analysis indicated increased thioredoxin-1, vascular endothelial cell growth factor, and ß-catenin levels in the ischemic wounds-thioredoxin-1 group compared with the ischemic wounds control and ischemic wounds-LacZ groups, as well as increased capillary density and cell proliferation, as represented by Ki-67 staining. CONCLUSION: Taken together, thioredoxin-1 gene therapy promotes vascular endothelial cell growth factor signaling and re-epithelialization and activates wound closure in mice with ischemic wounds.

Thioredoxin-1 attenuates sepsis-induced cardiomyopathy after cecal ligation and puncture in mice.

BACKGROUND: Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis. METHODS: Adult C57BL/6J and Trx-1Tg/+ mice were divided into wild-type sham (WTS), wild-type cecal ligation and puncture (WTCLP), Trx-1Tg/+sham (Trx-1Tg/+S), and Trx-1Tg/+CLP groups. Cardiac function was evaluated before surgery, 6 and 24 hours after CLP surgery. Immunohistochemical and Western blot analysis were performed after 24 hours in heart tissue sections. RESULTS: Echocardiography analysis showed preserved cardiac function in the Trx-1Tg/+ CLP group compared with the WTCLP group. Similarly, Western blot analysis revealed increased expression of Trx-1, heme oxygenase-1 (HO-1), survivin (an inhibitor of apoptosis [IAP] protein family), and decreased expression of thioredoxin-interacting protein (TXNIP), caspase-3, and 3- nitrotyrosine in the Trx-1Tg/+CLP group compared with the WTCLP group. Immunohistochemical analysis showed reduced 4-hydroxynonenal, apoptosis, and vascular leakage in the cardiac tissue of Trx-1Tg/+CLP mice compared with mice in the WTCLP group. CONCLUSIONS: Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia.

BACKGROUND: There is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. METHODS: To determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used-gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. RESULTS: Significant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. CONCLUSIONS: The overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

Regulation of A-Kinase-Anchoring Protein 12 by Heat Shock Protein A12B to Prevent Ventricular Dysfunction Following Acute Myocardial Infarction in Diabetic Rats.

We examined the effects of overexpressing HSPA12B on angiogenesis and myocardial function by intramyocardial administration of adenovirus encoding HSPA12B (Ad. HSPA12B) in a streptozotocin-induced diabetic rat subjected to myocardial infarction. Rats were divided randomly into six groups: control sham (CS) + Ad.LacZ, control myocardial infarction (CMI) + Ad.LacZ, control MI + Ad.HSPA12B, diabetic sham (DS) + Ad.LacZ, diabetic MI + Ad.LacZ and diabetic MI + Ad.HSPA12B. Following MI or sham surgery, the respective groups received either Ad.LacZ or Ad.HSPA12B via intramyocardial injections. We observed increased capillary and arteriolar density along with reduced fibrosis and preserved heart functions in DMI-AdHSPA12B compared to DMI-AdLacZ group. Western blot analysis demonstrated enhanced HSPA12B, vascular endothelial growth factor (VEGF), thioredoxin-1 (Trx-1) expression along with decreased expression of thioredoxin interacting protein (TXNIP) and A kinase anchoring protein 12 (AKAP12) in the DMI-AdHSPA12B compared to DMI-AdLacZ group. Our findings reveal that HSPA12B overexpression interacts with AKAP12 and downregulate TXNIP in diabetic rats following acute MI.

Increased survivability of ischemic skin flap tissue in Flk-1+/- mice by Pellino-1 intervention.

OBJECTIVE: Reduced skin flap survival due to ischemia is a serious concern during reconstructive cosmetic surgery. The absence of VEGF and its receptors during ischemia may lead to flap failure. We identified Peli1, a 46-kDa protein, as a proangiogenic molecule and is directly regulated by VEGF. Therefore, we hypothesized that Peli1 acts downstream of Flk-1/VEGFR2 and aids in skin flap survival during ischemia. METHODS: Scratch and matrigel assays were performed to observe cell proliferation, migration, and tube formation in vitro. Western blot analysis was carried out to detect the phosphorylation of Akt (p-Akt) and MAPKAPK2 (p-MK2) in HUVECs. The translational potential of Peli1 pretreatment in the rescue of skin flap tissue was studied in vivo using Flk-1+/- mice. Animals underwent dorsal ischemic skin flap surgery, and the tissue was collected on day 12 for analysis. RESULTS: Western blot analysis revealed a direct relationship between Peli1 and VEGF, as demonstrated by loss-of-function and gain-of-function studies. In addition, pretreatment with Ad.Peli1 restored the phosphorylation of Akt and MK2 and improved the migration potential of Flk-1-knockdown cells. Ad.Peli1 pretreatment salvaged the ischemic skin flap of Flk-1+/- mice by increasing blood perfusion and reducing the inflammatory response and the extent of necrosis. CONCLUSION: Our findings reveal that Peli1 is a proangiogenic molecule that acts downstream of VEGF-Flk-1 and restores angiogenesis and enhances skin flap survivability.

Protective effects of Phyllanthus emblicaagainst myocardial ischemia-reperfusion injury: the role of PI3-kinase/glycogen synthase kinase 3Beta/Beta-catenin pathway

Clinical studies of Phyllanthus emblica (P. emblica) have shown that it increases production of nitric oxide, glutathione, and high-density lipoprotein (HDL); decreases low-density lipoprotein (LDL), total cholesterol, triglycerides, and high-sensitivity C-reactive protein (hsCRP); and significantly inhibits platelet aggregation. The following study was designed to examine the effect of P. emblica treatment on myocardial ischemia-reperfusion (I/R) injury and identify the molecular targets and its underlying mechanism(s). Experimental animals were divided into four groups: control sham (CS), P. emblica sham (PS), control I/R (CIR), and P. emblica I/R (PIR). Rats in the P. emblica groups were gavaged with aqueous P. emblica solution (100 mg/kg body weight) for 30 days. After 30 days of gavaging, the I/R group underwent I/R surgery (45-min ischemia) followed by 4 or 30 days of reperfusion. Rats in the sham group underwent surgery without ligation. Left ventricular tissue samples, 4 and 30 days after I/R, were used for Western blot analysis and immunohistochemistry, respectively. Western blot analysis showed upregulation of phosphorylated Akt and GSK3-Alpha and increased nuclear translocation of Alpha-catenin in the PIR group versus CIR. PIR rats also indicated reduced 3-nitrotyrosine and Caspase-3 expression. Increased phosphorylation of endothelial nitric oxide synthase (p-eNOS) and upregulation of anti-apoptotic protein Bcl-2 were found in the PIR group. Echocardiography showed increased ejection fraction and fractional shortening and decreased left ventricular internal diameter in experimental subjects compared to controls. There was decreased fibrosis in P. emblica-treated rats compared to controls. The results of this study indicate that P. emblica is capable of upregulating the PI3K/Akt/AlphaGSK3/Alpha-catenin cardioprotective pathway, thereby preserving cardiac tissue during ischemia-reperfusion injury)

Thioredoxin-1 (Trx1) engineered mesenchymal stem cell therapy increased pro-angiogenic factors, reduced fibrosis and improved heart function in the infarcted rat myocardium.

INTRODUCTION: Engraftment of mesenchymal stem cells (MSCs) has emerged as a powerful candidate for mediating myocardial repair. In this study, we genetically modified MSCs with an adenovector encoding thioredoxin-1 (Ad.Trx1). Trx1 has been described as a growth regulator, a transcription factor regulator, a cofactor, and a powerful antioxidant. We explored whether engineered MSCs, when transplanted, are capable of improving cardiac function and angiogenesis in a rat model of myocardial infarction (MI). METHODS: Rat MSCs were cultured and divided into MSC, MSC+Ad.LacZ, and MSC+Ad.Trx1 groups. The cells were assayed for proliferation, and differentiation potential. In addition, rats were divided into control-sham (CS), control-MI (CMI), MSC+Ad.LacZ-MI (MLZMI), and MSC+Ad.Trx1-MI (MTrxMI) groups. MI was induced by left anterior descending coronary artery (LAD) ligation, and MSCs preconditioned with either Ad.LacZ or Ad.Trx1 were immediately administered to four sites in the peri-infarct zone. RESULTS: The MSC+Ad.Trx1 cells increased the proliferation capacity and maintained pluripotency, allowing them to divide into cardiomyocytes, smooth muscle, and endothelial cells. Western blot analysis, 4 days after treatment showed increased vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and C-X-C chemokine receptor type 4 (CXCR4). Also capillary density along with myocardial function as examined by echocardiography was found to be increased. Fibrosis was reduced in the MTrxMI group compared to MLZMI and CMI. Visualization of Connexin-43 by immunohistochemistry confirmed increased intercellular connections in the MTrxMI rats compared to MLZMI. CONCLUSION: Engineering MSCs to express Trx1 may prove to be a strategic therapeutic modality in the treatment of cardiac failure.

Protective effects of Phyllanthus emblica against myocardial ischemia-reperfusion injury: the role of PI3-kinase/glycogen synthase kinase 3ß/ß-catenin pathway.

Clinical studies of Phyllanthus emblica (P. emblica) have shown that it increases production of nitric oxide, glutathione, and high-density lipoprotein (HDL); decreases low-density lipoprotein (LDL), total cholesterol, triglycerides, and high-sensitivity C-reactive protein (hsCRP); and significantly inhibits platelet aggregation. The following study was designed to examine the effect of P. emblica treatment on myocardial ischemia-reperfusion (I/R) injury and identify the molecular targets and its underlying mechanism(s). Experimental animals were divided into four groups: control sham (CS), P. emblica sham (PS), control I/R (CIR), and P. emblica I/R (PIR). Rats in the P. emblica groups were gavaged with aqueous P. emblica solution (100 mg/kg body weight) for 30 days. After 30 days of gavaging, the I/R group underwent I/R surgery (45-min ischemia) followed by 4 or 30 days of reperfusion. Rats in the sham group underwent surgery without ligation. Left ventricular tissue samples, 4 and 30 days after I/R, were used for Western blot analysis and immunohistochemistry, respectively. Western blot analysis showed upregulation of phosphorylated Akt and GSK3-ß and increased nuclear translocation of ß-catenin in the PIR group versus CIR. PIR rats also indicated reduced 3-nitrotyrosine and Caspase-3 expression. Increased phosphorylation of endothelial nitric oxide synthase (p-eNOS) and upregulation of anti-apoptotic protein Bcl-2 were found in the PIR group. Echocardiography showed increased ejection fraction and fractional shortening and decreased left ventricular internal diameter in experimental subjects compared to controls. There was decreased fibrosis in P. emblica-treated rats compared to controls. The results of this study indicate that P. emblica is capable of upregulating the PI3K/Akt/GSK3ß/ß-catenin cardioprotective pathway, thereby preserving cardiac tissue during ischemia-reperfusion injury.