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.

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.

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.

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.

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.

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.

Cardiomyopathy and Worsened Ischemic Heart Failure in SM22-α Cre-Mediated Neuropilin-1 Null Mice: Dysregulation of PGC1α and Mitochondrial Homeostasis.

OBJECTIVE: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive. APPROACH AND RESULTS: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis. CONCLUSIONS: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.

Deletion of prolyl hydroxylase domain proteins (PHD1, PHD3) stabilizes hypoxia inducible factor-1 alpha, promotes neovascularization, and improves perfusion in a murine model of hind-limb ischemia.

BACKGROUND: There is an emerging focus on investigating innovative therapeutic molecules that can potentially augment neovascularization in order to treat peripheral arterial disease (PAD). Although prolyl hydroxylase domain proteins 1 and 3 (PHD1 and PHD3) may modulate angiogenesis via regulation of hypoxia inducible factor-1α (HIF-1α), there has been no study directly addressing their roles in ischemia-induced vascular growth. We hypothesize that PHD1(-/-) or PHD3(-/-) deficiency might promote angiogenesis in the murine hind-limb ischemia (HLI) model. STUDY DESIGN: Wild type (WT), PHD1(-/-) and PHD3(-/-) male mice aged 8-12weeks underwent right femoral artery ligation. Post-procedurally, motor function assessment and laser Doppler imaging were periodically performed. The mice were euthanized after 28days and muscles were harvested. Immunohistochemical analysis was performed to determine the extent of angiogenesis by measuring capillary and arteriolar density. VEGF expression was quantified by enzyme-linked immunosorbent assay (ELISA). Bcl-2 and HIF-1α were analyzed by immunofluorescence. Fibrosis was measured by picrosirius red staining. RESULTS: PHD1(-/-) and PHD3(-/-) mice showed significantly improved recovery of perfusion and motor function score when compared to WT after femoral artery ligation. These mice also exhibited increased capillary and arteriolar density, capillary/myocyte ratio along with decreased fibrosis compared to WT. VEGF, Bcl-2 and HIF-1α expression increased in PHD1(-/-) and PHD3(-/-) mice compared to WT. CONCLUSIONS: Taken together these results suggest that PHD1 and PHD3 deletions promote angiogenesis in ischemia-injured tissue, and may present a promising therapeutic strategy in treating PAD.

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.

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.

Novel role of NADPH oxidase in ischemic myocardium: a study with Nox2 knockout mice.

Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30 min of ischemia followed by 2 h of reperfusion (IR) or IP prior to 30 min ischemia and 2 h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63 mmHg), dp/dt (max) (893 versus 1,027 mmHg/s), and aortic flow (0.9 versus 1.8 ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120 min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as ß-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.

HIF-prolyl hydroxylases and cardiovascular diseases.

Prolyl hydroxylases belong to the family of iron- and 2-oxoglutamate-dependent dioxygenase enzyme. Several distinct prolyl hydroxylases have been identified. The hypoxia-inducible factor (HIF) prolyl hydroxylase termed prolyl hydroxylase domain (PHD) enzymes play an important role in oxygen regulation in the physiological network. There are three isoforms that have been identified: PHD1, PHD2 and PHD3. Deletion of PHD enzymes result in stabilization of HIFs and offers potential treatment options for many ischemic disorders such as peripheral arterial occlusive disease, myocardial infarction, and stroke. All three isoforms are oxygen sensors that regulate the stability of HIFs. The degradation of HIF-1α is regulated by hydroxylation of the 402/504 proline residue by PHDs. Under hypoxic conditions, lack of oxygen causes hydroxylation to cease HIF-1α stabilization and subsequent translocation to the nucleus where it heterodimerizes with the constitutively expressed ß subunit. Binding of the HIF-heterodimer to specific DNA sequences, named hypoxia-responsive elements, triggers the transactivation of target genes. PHD regulation of HIF-1α-mediated cardioprotection has resulted in considerable interest in these molecules as potential therapeutic targets in cardiovascular and ischemic diseases. In recent years, attention has been directed towards identifying small molecule inhibitors of PHD. It is postulated that such inhibition might lead to a clinically useful strategy for protecting the myocardium against ischemia and reperfusion injury. Recently, it has been reported that the orally absorbed PHD inhibitor GSK360A can modulate HIF-1α signaling and protect the failing heart following myocardial infarction. Furthermore, PHD1 deletion has been found to have beneficial effects through an increase in tolerance to hypoxia of skeletal muscle by reprogramming basal metabolism. In the mouse liver, such deletion has resulted in protection against ischemia and reperfusion. As a result of these preliminary findings, PHDs is attracting increasing interest as potential therapeutic targets in a wide range of diseases.

Safety of oligonol, a highly bioavailable lychee-derived polyphenolic antioxidant, on liver, kidney and heart function in rats.

Oligonol (OLG), derived from lychee fruit, is a novel compound produced from the oligomerization of polyphenols. In this study, the acute effect of OLG treatment was investigated on heart, liver and kidney in rats. OLG treatment at two different doses (15 or 30 mg/kg body weight) and two different time points (1 day or 7 days of treatment) demonstrated that no toxic effects were observed on heart, liver and renal functions. Moreover, OLG did not induce any DNA damage or oxidative stress as measured by 8-hydroxy-2'-deoxyguanosine levels in plasma. OLG supplementation increased the phosphorylation of myocardial endothelial nitric oxide (NO) level (p-eNOS) in both the treatment groups. Even the low dose OLG treatment (15mg/kg b.w) demonstrated an increase in p-eNOS/eNOS ratio after normalization of p-eNOS values with eNOS on day 1 (1.5-fold) and day 7 (2.2-fold) groups as compared to control. The above results suggest that OLG treatment increases endothelial NO levels and may play a role in NO-mediated vasodilatory effects without adverse side effects on cardiovascular function. This endothelial NO production may underlie the beneficial effect of OLG in cardiovascular health.

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.

Thioredoxin 1 enhances neovascularization and reduces ventricular remodeling during chronic myocardial infarction: a study using thioredoxin 1 transgenic mice.

Oxidative stress plays a crucial role in disruption of neovascularization by alterations in thioredoxin 1 (Trx1) expression and its interaction with other proteins after myocardial infarction (MI). We previously showed that Trx1 has angiogenic properties, but the possible therapeutic significance of overexpressing Trx1 in chronic MI has not been elucidated. Therefore, we explored the angiogenic and cardioprotective potential of Trx1 in an in vivo MI model using transgenic mice overexpressing Trx1. Wild-type (W) and Trx1 transgenic (Trx1(Tg/+)) mice were randomized into W sham (WS), Trx1(Tg/+) sham (TS), WMI, and TMI. MI was induced by permanent occlusion of LAD coronary artery. Hearts from mice overexpressing Trx1 exhibited reduced fibrosis and oxidative stress and attenuated cardiomyocyte apoptosis along with increased vessel formation compared to WMI. We found significant inhibition of Trx1 regulating proteins, TXNIP and AKAP 12, and increased p-Akt, p-eNOS, p-GSK-3ß, HIF-1α, ß-catenin, VEGF, Bcl-2, and survivin expression in TMI compared to WMI. Echocardiography performed 30days after MI revealed significant improvement in myocardial functions in TMI compared to WMI. Our study identifies a potential role for Trx1 overexpression and its association with its regulatory proteins TXNIP, AKAP12, and subsequent activation of Akt/GSK-3ß/ß-catenin/HIF-1α-mediated VEGF and eNOS expression in inducing angiogenesis and reduced ventricular remodeling. Hence, Trx1 and other proteins identified in our study may prove to be potential therapeutic targets in the treatment of ischemic heart disease.

Postischemic deactivation of cardiac aldose reductase: role of glutathione S-transferase P and glutaredoxin in regeneration of reduced thiols from sulfenic acids.

Aldose reductase (AR) is a multifunctional enzyme that catalyzes the reduction of glucose and lipid peroxidation-derived aldehydes. During myocardial ischemia, the activity of AR is increased due to the oxidation of its cysteine residues to sulfenic acids. It is not known, however, whether the activated, sulfenic form of the protein (AR-SOH) is converted back to its reduced, unactivated state (AR-SH). We report here that in perfused mouse hearts activation of AR during 15 min of global ischemia is completely reversed by 30 min of reperfusion. During reperfusion, AR-SOH was converted to a mixed disulfide (AR-SSG). Deactivation of AR and the appearance of AR-SSG during reperfusion were delayed in hearts of mice lacking glutathione S-transferase P (GSTP). In vitro, GSTP accelerated glutathiolation and inactivation of AR-SOH. Reduction of AR-SSG to AR-SH was facilitated by glutaredoxin (GRX). Ischemic activation of AR was increased in GRX-null hearts but was attenuated in the hearts of cardiospecific GRX transgenic mice. Incubation of AR-SSG with GRX led to the regeneration of the reduced form of the enzyme. In ischemic cardiospecific AR transgenic hearts, AR was co-immunoprecipitated with GSTP, whereas in reperfused hearts, the association of AR with GRX was increased. These findings suggest that upon reperfusion of the ischemic heart AR-SOH is converted to AR-SSG via GSTP-assisted glutathiolation. AR-SSG is then reduced by GRX to AR-SH. Sequential catalysis by GSTP and GRX may be a general redox switching mechanism that regulates the reduction of protein sulfenic acids to cysteines.

Thioredoxin-1 gene therapy enhances angiogenic signaling and reduces ventricular remodeling in infarcted myocardium of diabetic rats.

BACKGROUND: The present study evaluated the reversal of diabetes-mediated impairment of angiogenesis in a myocardial infarction model of type 1 diabetic rats by intramyocardial administration of an adenoviral vector encoding thioredoxin-1 (Ad.Trx1). Various studies have linked diabetes-mediated impairment of angiogenesis to dysfunctional antioxidant systems in which thioredoxin-1 plays a central role. METHODS AND RESULTS: Ad.Trx1 was administered intramyocardially in nondiabetic and diabetic rats immediately after myocardial infarction. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. Myocardial function was measured by echocardiography 30 days after the intervention. The Ad.Trx1-administered group exhibited reduced fibrosis, oxidative stress, and cardiomyocyte and endothelial cell apoptosis compared with the diabetic myocardial infarction group, along with increased capillary and arteriolar density. Western blot and immunohistochemical analysis demonstrated myocardial overexpression of thioredoxin-1, heme oxygenase-1, vascular endothelial growth factor, and p38 mitogen-activated protein kinase-beta, as well as decreased phosphorylated JNK and p38 mitogen-activated protein kinase-alpha, in the Ad.Trx1-treated diabetic group. Conversely, we observed a significant reduction in the expression of vascular endothelial growth factor in nondiabetic and diabetic animals treated with tin protoporphyrin (SnPP, a heme oxygenase-1 enzyme inhibitor), even after Ad.Trx1 therapy. Echocardiographic analysis after 4 weeks of myocardial infarction revealed significant improvement in myocardial functional parameters such as ejection fraction, fractional shortening, and E/A ratio in the Ad.Trx1-administered group compared with the diabetic myocardial infarction group. CONCLUSIONS: This study demonstrates for the first time that impairment of angiogenesis and myocardial dysfunction can be regulated by Ad.Trx1 gene therapy in streptozotocin-induced diabetic rats subjected to infarction.

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.

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.