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1.
Int J Obes (Lond) ; 2021 Oct 08.
Article in English | MEDLINE | ID: mdl-34625660

ABSTRACT

Obesity is a risk factor for coronavirus disease 2019 (COVID-19) infection, with studies demonstrating the prevalence of individuals with obesity admitted with COVID-19 ranging between 30 and 60%. We determined whether early changes in microRNAs (miRNAs) are associated with dysregulation of angiotensin-converting enzyme 2 (ACE2), the specific functional receptor for severe acute respiratory syndrome coronavirus 2. ACE2 is a membrane-bound enzyme that catalyzes the conversion of angiotensin II to angiotensin 1-7 the latter having cardioprotective and vasorelaxation effects. Quantitative real-time PCR analysis of plasma samples for circulating miRNAs showed upregulation of miR-200c and miR-let-7b in otherwise healthy individuals with obesity. This was associated with significant downregulation of ACE2, a direct target for both miRNAs, in individuals with obesity. Correlation analysis confirmed a significant negative correlation between ACE2 and both the miRNAs. Studies showed that despite being the functional receptor, inhibition/downregulation of ACE2 did not reduce the severity of COVID-19 infection. In contrast, increased angiotensin II following inhibition of ACE2 may increase the severity of the disease. Taken together, our novel results identify that upregulation of miR-200c may increase the susceptibility of individuals with obesity to COVID-19. Considering miRNA are the earliest molecular regulators, the level of circulating miR-200c could be a potential biomarker in the early identification of those at the risk of severe COVID-19.

2.
Int J Cardiol ; 341: 70-73, 2021 Oct 15.
Article in English | MEDLINE | ID: mdl-34461161

ABSTRACT

BACKGROUND: The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem cells. However, the relative complexity of non-resident cardiac progenitor cell (CPC) therapy makes modulation of resident CPCs a more attractive treatment target. Thiamine analogues improve resident CPC function in pre-clinical models. In this double blinded randomised controlled trial (identifier: ACTRN12614000755639), we examined whether thiamine would improve CPC function in humans. METHODS AND RESULTS: High dose oral thiamine (one gram twice daily) or matching placebo was administered 3-5 days prior to coronary artery bypass surgery (CABG). Right atrial appendages were collected at the time of CABG, and CPCs isolated. There was no difference in the primary outcome (proliferation ability of CPCs) between treatment groups. Older age was not associated with decreased proliferation ability. In exploratory analyses, isolated CPCs in the thiamine group showed an increase in the proportion of CD34-/CD105+ (endoglin) cells, but no difference in CD34-/CD90+ or CD34+ cells. Thiamine increased maximum force developed by isolated trabeculae, with no difference in relaxation time or beta-adrenergic responsiveness. CONCLUSION: Thiamine does not improve proliferation ability of CPC in patients undergoing CABG, but increases the proportion of CD34-/CD105+ cells. Having not met its primary endpoint, this study provides the impetus to re-examine CPC biology prior to any clinical outcome-based trial examining potential beneficial cardiovascular effects of thiamine.

3.
J Manipulative Physiol Ther ; 44(5): 420-431, 2021 06.
Article in English | MEDLINE | ID: mdl-34376321

ABSTRACT

OBJECTIVE: The purpose of the present study was to determine the neuroendocrine response after a thoracic spinal manipulation in people with Achilles tendinopathy. METHODS: This was a randomized 2-sequence, 2-period crossover trial. A total of 24 participants, mean (standard deviation) age of 48 (7) years, with a diagnosis of Achilles tendinopathy (>3 mo) were randomly assigned into sequence 1 (sham intervention and then thoracic spinal manipulation) or sequence 2 (thoracic spinal manipulation and then sham intervention). The trial was conducted at a university laboratory with a washout period of 1 week. The primary outcome measure was the testosterone/cortisol (T/C) ratio (salivary samples). The secondary outcome measures included heart rate variability (measured with electrocardiography) and total oxygenation index (nmol/L) of calf muscle and Achilles tendon (measured with near-infrared spectroscopy). A 2-way mixed-model analysis of variance was performed. The statistic of interest was the condition by time interaction. RESULTS: A statistically significant condition by time interaction was found for the T/C ratio (mean difference: -0.16; confidence interval: -0.33 to 0.006; interaction: P < .05) and the total oxygenation index (mean difference: 1.35; confidence interval: -1.3 to 4.1; interaction: P < .05) of calf muscle but not for Achilles tendon (P = .6); however, no difference was found for heart rate variability (P = .5). CONCLUSION: In people with Achilles tendinopathy, thoracic spinal manipulation resulted in immediate increase in the total oxygenation index in the calf muscle followed by an increase in the T/C ratio 6 hours post-intervention.


Subject(s)
Achilles Tendon , Manipulation, Spinal , Tendinopathy , Cross-Over Studies , Humans , Middle Aged , Muscle, Skeletal , Tendinopathy/therapy
4.
Article in English | MEDLINE | ID: mdl-34011169

ABSTRACT

Significance: Diabetic heart disease (DHD) is the primary cause of mortality in people with diabetes. A significant contributor to the development of DHD is the disruption of redox balance due to reactive oxygen species (ROS) overproduction resulting from sustained high glucose levels. Therapies specifically focusing on the suppression of ROS will hugely benefit patients with DHD. Recent Advances: In addition to the gold standard pharmacological therapies, the recent development of gene therapy provides an exciting avenue for developing new therapeutics to treat ROS-mediated DHD. In particular, microRNAs (miRNAs) are gaining interest due to their crucial role in several physiological and pathological processes, including DHD. Critical Issues: miRNAs have many targets and differential function depending on the environment. Therefore, a proper understanding of the function of miRNAs in specific cell types and cell states is required for the successful application of this technology. In the present review, we first provide an overview of the role of ROS in contributing to DHD and the currently available treatments. We then discuss the newer gene therapies with a specific focus on the role of miRNAs as the causative factors and therapeutic targets to combat ROS-mediated DHD. Future Directions: The future of miRNA therapeutics in tackling ROS-mediated DHD is dependent on a complete understanding of how miRNAs behave in different cells and environments. Future research should also aim to develop conditional miRNA therapeutic platforms capable of switching on and off in response to disruptions in the redox state.

6.
Diabetologia ; 64(6): 1422-1435, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33655378

ABSTRACT

AIMS/HYPOTHESIS: Diabetes mellitus causes a progressive loss of functional efficacy in stem cells, including cardiac progenitor cells (CPCs). The underlying molecular mechanism is still not known. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate genes at the post-transcriptional level. We aimed to determine if diabetes mellitus induces dysregulation of miRNAs in CPCs and to test if in vitro therapeutic modulation of miRNAs would improve the functions of diabetic CPCs. METHODS: CPCs were isolated from a mouse model of type 2 diabetes (db/db), non-diabetic mice and human right atrial appendage heart tissue. Total RNA isolated from mouse CPCs was miRNA profiled using Nanostring analysis. Bioinformatic analysis was employed to predict the functional effects of altered miRNAs. MS analysis was applied to determine the targets, which were confirmed by western blot analysis. Finally, to assess the beneficial effects of therapeutic modulation of miRNAs in vitro and in vivo, prosurvival miR-30c-5p was overexpressed in mouse and human diabetic CPCs, and the functional consequences were determined by measuring the level of apoptotic cell death, cardiac function and mitochondrial membrane potential (MMP). RESULTS: Among 599 miRNAs analysed in mouse CPCs via Nanostring analysis, 16 miRNAs showed significant dysregulation in the diabetic CPCs. Using bioinformatics tools and quantitative real-time PCR (qPCR) validation, four altered miRNAs (miR-30c-5p, miR-329-3p, miR-376c-3p and miR-495-3p) were identified to play an important role in cell proliferation and survival. Diabetes mellitus significantly downregulated miR-30c-5p, while it upregulated miR-329-3p, miR-376c-3p and miR-495-3p. MS analysis revealed proapoptotic voltage-dependent anion-selective channel 1 (VDAC1) as a direct target for miR-30c-5p, and cell cycle regulator, cyclin-dependent protein kinase 6 (CDK6), as the direct target for miR-329-3p, miR-376c-3p and miR-495-3p. Western blot analyses showed a marked increase in VDAC1 expression, while CDK6 expression was downregulated in diabetic CPCs. Finally, in vitro and in vivo overexpression of miR-30c-5p markedly reduced the apoptotic cell death and preserved MMP in diabetic CPCs via inhibition of VDAC1. CONCLUSIONS/INTERPRETATION: Our results demonstrate that diabetes mellitus induces a marked dysregulation of miRNAs associated with stem cell survival, proliferation and differentiation, and that therapeutic overexpression of prosurvival miR-30c-5p reduced diabetes-induced cell death and loss of MMP in CPCs via the newly identified target for miR-30c-5p, VDAC1.

7.
Cardiovasc Diabetol ; 20(1): 50, 2021 02 22.
Article in English | MEDLINE | ID: mdl-33618724

ABSTRACT

BACKGROUND: Acetylcholine (ACh) plays a crucial role in the function of the heart. Recent evidence suggests that cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that comprises of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and type-2 muscarinic ACh receptors (M2AChR) to synthesize, release, degrade ACh as well as for ACh to transduce a signal. NNCS is linked to cardiac cell survival, angiogenesis and glucose metabolism. Impairment of these functions are hallmarks of diabetic heart disease (DHD). The role of the NNCS in DHD is unknown. The aim of this study was to examine the effect of diabetes on cardiac NNCS and determine if activation of cardiac NNCS is beneficial to the diabetic heart. METHODS: Ventricular samples from type-2 diabetic humans and db/db mice were used to measure the expression pattern of NNCS components (ChAT, CHT1, VAChT, AChE and M2AChR) and glucose transporter-4 (GLUT-4) by western blot analysis. To determine the function of the cardiac NNCS in the diabetic heart, a db/db mouse model with cardiac-specific overexpression of ChAT gene was generated (db/db-ChAT-tg). Animals were followed up serially and samples collected at different time points for molecular and histological analysis of cardiac NNCS components and prosurvival and proangiogenic signaling pathways. RESULTS: Immunoblot analysis revealed alterations in the components of cardiac NNCS and GLUT-4 in the type-2 diabetic human and db/db mouse hearts. Interestingly, the dysregulation of cardiac NNCS was followed by the downregulation of GLUT-4 in the db/db mouse heart. Db/db-ChAT-tg mice exhibited preserved cardiac and vascular function in comparison to db/db mice. The improved function was associated with increased cardiac ACh and glucose content, sustained angiogenesis and reduced fibrosis. These beneficial effects were associated with upregulation of the PI3K/Akt/HIF1α signaling pathway, and increased expression of its downstream targets-GLUT-4 and VEGF-A. CONCLUSION: We provide the first evidence for dysregulation of the cardiac NNCS in DHD. Increased cardiac ACh is beneficial and a potential new therapeutic strategy to prevent or delay the development of DHD.


Subject(s)
Acetylcholine/metabolism , Diabetes Mellitus, Type 2/complications , Diabetic Cardiomyopathies/prevention & control , Glucose/metabolism , Heart Ventricles/metabolism , Acetylcholinesterase/metabolism , Aged , Animals , Case-Control Studies , Choline O-Acetyltransferase/genetics , Choline O-Acetyltransferase/metabolism , Diabetes Mellitus, Type 2/metabolism , Diabetic Cardiomyopathies/etiology , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/physiopathology , Disease Models, Animal , Female , GPI-Linked Proteins/metabolism , Glucose Transporter Type 4/metabolism , Humans , Male , Membrane Transport Proteins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Receptor, Muscarinic M2/metabolism , Symporters/metabolism , Vesicular Acetylcholine Transport Proteins/metabolism
8.
Nutr Diabetes ; 11(1): 8, 2021 02 08.
Article in English | MEDLINE | ID: mdl-33558456

ABSTRACT

Diabetes is associated with cardiac metabolic disturbances and increased heart failure risk. Plasma fructose levels are elevated in diabetic patients. A direct role for fructose involvement in diabetic heart pathology has not been investigated. The goals of this study were to clinically evaluate links between myocardial fructose and sorbitol (a polyol pathway fructose precursor) levels with evidence of cardiac dysfunction, and to experimentally assess the cardiomyocyte mechanisms involved in mediating the metabolic effects of elevated fructose. Fructose and sorbitol levels were increased in right atrial appendage tissues of type 2 diabetic patients (2.8- and 1.5-fold increase respectively). Elevated cardiac fructose levels were confirmed in type 2 diabetic rats. Diastolic dysfunction (increased E/e', echocardiography) was significantly correlated with cardiac sorbitol levels. Elevated myocardial mRNA expression of the fructose-specific transporter, Glut5 (43% increase), and the key fructose-metabolizing enzyme, Fructokinase-A (50% increase) was observed in type 2 diabetic rats (Zucker diabetic fatty rat). In neonatal rat ventricular myocytes, fructose increased glycolytic capacity and cytosolic lipid inclusions (28% increase in lipid droplets/cell). This study provides the first evidence that elevated myocardial fructose and sorbitol are associated with diastolic dysfunction in diabetic patients. Experimental evidence suggests that fructose promotes the formation of cardiomyocyte cytosolic lipid inclusions, and may contribute to lipotoxicity in the diabetic heart.


Subject(s)
Diabetes Mellitus, Type 2/pathology , Fructose/analysis , Lipid Metabolism , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Sorbitol/analysis , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Fructokinases , Fructose/metabolism , Glucose/metabolism , Humans , Lipid Droplets/metabolism , Male , Myocardium/chemistry , Rats , Rats, Zucker , Sorbitol/metabolism , Ventricular Dysfunction, Left/pathology
9.
Int J Pharm ; 597: 120345, 2021 Mar 15.
Article in English | MEDLINE | ID: mdl-33545287

ABSTRACT

Inhaled delivery of rifampicin has the potential to achieve high drug concentrations in the lung and the blood for efficient treatment of tuberculosis (TB). Due to its existence as polymorphs, in vivo evaluation of the respiratory tract safety of inhalable amorphous and crystalline rifampicin particles, at clinically relevant high-dose, is necessary. This study investigates the lung and liver safety and the tissue distribution of rifampicin after intra-tracheal administration of high (≥25 mg/kg) doses of amorphous and crystalline powder formulations to Sprague Dawley rats. Powder formulations were administered by intra-tracheal insufflation to rats. Lung and liver safety were evaluated by histopathology. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) assays were performed to study the hepatic effects. Rifampicin was quantified in the tissues using LC-MS/MS. Intra-tracheal administration of rifampicin decreased the drug burden on the liver compared to oral administration based on its lower serum ALT activity. Repeated-dose intra-tracheal rifampicin was well tolerated by rats, confirmed by the absence of drug or delivery induced complexities. The histopathological evaluation of rat lungs, after both single and repeated drug administration for seven days, suggested the absence of drug-induced toxicity. Following single intra-tracheal delivery of 50 mg/kg doses, comparable rifampicin concentrations to that from same oral dose were observed in lung, liver, heart and brain. Inhaled delivery of high-dose rifampicin was safe to rat lungs and liver suggesting its potential for localized as well as systemic drug delivery without toxicity concerns.


Subject(s)
Rifampin , Tandem Mass Spectrometry , Administration, Inhalation , Animals , Chromatography, Liquid , Lung , Powders , Rats , Rats, Sprague-Dawley , Rifampin/adverse effects , Tissue Distribution
10.
Eur J Pharm Biopharm ; 162: 1-11, 2021 May.
Article in English | MEDLINE | ID: mdl-33639255

ABSTRACT

Rifampicin is one of the key drugs used to treat tuberculosis and is currently used orally. The use of higher oral doses of rifampicin is desired for better therapeutic efficacy, but this is accompanied by increased risk of systemic toxicity thus limiting its recommended oral dose to 10 mg/kg per day. Inhaled delivery of rifampicin is a potential alternative mode of delivery, to achieve high drug concentrations in both the lung and potentially the systemic circulation. In addition, rifampicin exists either as amorphous or crystalline particles, which may show different pharmacokinetic behaviour. However, disposition behaviour of amorphous and crystalline rifampicin formulations after inhaled high-dose delivery is unknown. In this study, rifampicin pharmacokinetics after intra-tracheal administration of carrier-free, amorphous and crystalline powder formulations to Sprague Dawley rats were evaluated. The formulations were administered once daily for seven days by oral, intra-tracheal and oral plus intra-tracheal delivery, and the pharmacokinetics were studied on day 0 and day 6. Intra-tracheal administration of the amorphous formulation resulted in a higher area under the plasma concentration curve (AUC) compared to the crystalline formulation. For both formulations, the intra-tracheal delivery led to significantly higher AUC compared to the oral delivery at the same dose suggesting higher rifampicin bioavailability from the inhaled route. Increasing the intra-tracheal dose resulted in a more than dose proportional AUC suggesting non-linear pharmacokinetics of rifampicin from the inhaled route. Upon repeated administration for seven days, no significant decrease in the AUCs were observed suggesting the absence of rifampicin induced enzyme auto-induction in this study. The present study suggests an advantage of inhaled delivery of rifampicin in achieving higher drug bioavailability compared to the oral route.

11.
Antioxid Redox Signal ; 34(15): 1151-1164, 2021 May 20.
Article in English | MEDLINE | ID: mdl-33226850

ABSTRACT

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.

12.
Cell Transplant ; 29: 963689720972328, 2020.
Article in English | MEDLINE | ID: mdl-33153286

ABSTRACT

Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death worldwide. Regenerative medicine with stem cell therapy is a promising tool for cardiac repair. Combination of different cell types has been shown to improve the therapeutic potential, which is thought to be due to synergistic or complimentary reparative effects. We investigated if the combination of cardiac progenitor cells (CPCs) of right atrial appendage (RAA) and left ventricle (LV) that are isolated from the same patient exert synergistic or complimentary paracrine effects for apoptotic cell death and angiogenesis in an in vitro model. Flow cytometry analysis showed that both RAA and LV CPCs expressed the mesenchymal cell markers CD90 and CD105, and were predominantly negative for the hematopoietic cell marker, CD34. Analysis of conditioned media (CM) collected from the CPCs cultured either alone or in combination in serum-deprived hypoxic conditions to simulate ischemia showed marked increase in the level of pro-survival hepatocyte growth factor and pro-angiogenic vascular endothelial growth factor-A in the combined RAA and LV CPC group. Next, to determine the therapeutic potential of CM, AC16 human ventricular cardiomyocytes and human umbilical vein endothelial cells (HUVECs) were treated with CM. Results showed a significant reduction in hypoxia-induced apoptosis of human cardiomyocytes treated with CM collected from combined RAA and LV CPC group. Similarly, matrigel assay showed a significantly increased tube length formed by HUVECs when treated with CM from combined RAA and LV CPC group. Our study provided evidence that the combination of RAA CPCs and LV CPCs may have superior therapeutic effects due to synergistic paracrine effects for cardiac repair. Therefore, in vivo studies are warranted to determine if a combination of different stem cell types have greater therapeutic potential than single-cell therapies.

14.
Circ Res ; 127(11): 1384-1400, 2020 11 06.
Article in English | MEDLINE | ID: mdl-32907486

ABSTRACT

RATIONALE: Diabetic heart disease (DHD) is a debilitating manifestation of type 2 diabetes mellitus. Exercise has been proposed as a potential therapy for DHD, although the effectiveness of exercise in preventing or reversing the progression of DHD remains controversial. Cardiac function is critically dependent on the preservation of coronary vascular function. OBJECTIVE: We aimed to elucidate the effectiveness and mechanisms by which exercise facilitates coronary and cardiac-protection during the onset and progression of DHD. METHODS AND RESULTS: Diabetic db/db and nondiabetic mice, with or without underlying cardiac dysfunction (16 and 8 weeks old, respectively) were subjected to either moderate-intensity exercise or high-intensity exercise for 8 weeks. Subsequently, synchrotron microangiography, immunohistochemistry, Western blot, and real-time polymerase chain reaction were used to assess time-dependent changes in cardiac and coronary structure and function associated with diabetes mellitus and exercise and determine whether these changes reflect the observed changes in cardiac-enriched and vascular-enriched microRNAs (miRNAs). We show that, if exercise is initiated from 8 weeks of age, both moderate-intensity exercise and high-intensity exercise prevented the onset of coronary and cardiac dysfunction, apoptosis, fibrosis, microvascular rarefaction, and disruption of miRNA signaling, as seen in the nonexercised diabetic mice. Conversely, the cardiovascular benefits of moderate-intensity exercise were absent if the exercise was initiated after the diabetic mice had already established cardiac dysfunction (ie, from 16 weeks of age). The experimental silencing or upregulation of miRNA-126 activity suggests the mechanism underpinning the cardiovascular benefits of exercise were mediated, at least in part, through tissue-specific miRNAs. CONCLUSIONS: Our findings provide the first experimental evidence for the critical importance of early exercise intervention in ameliorating the onset and progression of DHD. Our results also suggest that the beneficial effects of exercise are mediated through the normalization of cardiovascular-enriched miRNAs, which are dysregulated in DHD.


Subject(s)
Diabetes Mellitus, Type 2/therapy , Diabetic Cardiomyopathies/prevention & control , Exercise Therapy , MicroRNAs/metabolism , Myocardium/metabolism , Physical Conditioning, Animal , Animals , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/physiopathology , Diabetic Cardiomyopathies/genetics , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/physiopathology , Disease Models, Animal , Female , Fibrosis , Gene Expression Regulation , Male , Mice , MicroRNAs/genetics , Myocardium/pathology , Running , Signal Transduction , Time Factors , Ventricular Function, Left , Ventricular Remodeling
15.
Sci Rep ; 10(1): 13651, 2020 08 12.
Article in English | MEDLINE | ID: mdl-32788622

ABSTRACT

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD). Yet, the mechanistic link between diabetes and PAD remains unclear. This study proposes that dysregulation of the endogenous hormone ghrelin, a potent modulator of vascular function, underpins the causal link between diabetes and PAD. Moreover, this study aimed to demonstrate the therapeutic potential of exogenous ghrelin in a diabetic mouse model of PAD. Standard ELISA analysis was used to quantify and compare circulating levels of ghrelin between (i) human diabetic patients with or without PAD (clinic) and (ii) db/db diabetic and non-diabetic mice (lab). Db/db mice underwent unilateral hindlimb ischaemia (HLI) for 14 days and treated with or without exogenous ghrelin (150 µg/kg/day.) Subsequently vascular reparation, angiogenesis, hindlimb perfusion, structure and function were assessed using laser Doppler imaging, micro-CT, microangiography, and protein and micro-RNA (miRNA) analysis. We further examined hindlimb perfusion recovery of ghrelin KO mice to determine whether an impaired vascular response to HLI is linked to ghrelin dysregulation in diabetes. Patients with PAD, with or without diabetes, had significantly lower circulating levels of endogenous ghrelin, compared to healthy individuals. Diabetic db/db mice had ghrelin levels that were only 7% of non-diabetic mice. The vascular reparative capacity of diabetic db/db mice in response to HLI was impaired compared to non-diabetic mice and, importantly, comparable to ghrelin KO mice. Daily therapeutic treatment of db/db mice with ghrelin for 14 days post HLI, stimulated angiogenesis, and improved skeletal muscle architecture and cell survival, which was associated with an increase in pro-angiogenic miRNAs-126 and -132. These findings unmask an important role for endogenous ghrelin in vascular repair following limb ischemia, which appears to be downregulated in diabetic patients. Moreover, these results implicate exogenous ghrelin as a potential novel therapy to enhance perfusion in patients with lower limb PAD, especially in diabetics.


Subject(s)
Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Ghrelin/metabolism , Hindlimb/pathology , Ischemia/complications , Neovascularization, Pathologic/pathology , Peripheral Arterial Disease/pathology , Aged , Animals , Case-Control Studies , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Female , Humans , Male , Mice , MicroRNAs/genetics , Middle Aged , Neovascularization, Pathologic/etiology , Neovascularization, Pathologic/metabolism , Peripheral Arterial Disease/complications , Peripheral Arterial Disease/metabolism
16.
Int J Pharm ; 587: 119602, 2020 Sep 25.
Article in English | MEDLINE | ID: mdl-32663580

ABSTRACT

Rifampicin is a first-line, highly effective drug currently used orally as a part of a lengthy multi-drug regimen against tuberculosis (TB). Despite the potential of inhaled therapy as an effective approach for TB treatment, an inhalable formulation of rifampicin has not yet been developed for clinical use. In order to do so, it is necessary to evaluate its solid-state properties, which regulate important characteristics like solubility, dissolution, aerosolization, stability and bioavailability. In this study, a crystallization technique and spray drying were used to prepare inhalable rifampicin formulations. Spray drying yielded amorphous formulation of rifampicin while crystalline dihydrate and pentahydrate formulations were obtained by crystallization. The powders were evaluated for their solid-state properties, in vitro aerosolization and aerosolization stability for three months when stored at different relative humidity conditions. All formulations had a mean particle size smaller than 3.8 µm and had a fine particle fraction (FPF) higher than 58.0%. Amorphous and crystalline dihydrate formulations showed no change in aerosolization parameters (emitted dose and FPF) upon storage for three months. The amorphous and pentahydrate formulations were found to undergo oxidative degradation upon storage, and a decrease in their drug content was observed. Among the formulations prepared, rifampicin dihydrate formulation showed the least degradation over the three months period. The inhalable rifampicin formulations prepared in this study, being excipient free, have the potential to be delivered as inhaled dry powder high-dose rifampicin to the lungs for effective treatment of TB.


Subject(s)
Dry Powder Inhalers , Rifampin , Administration, Inhalation , Aerosols , Particle Size , Powders
17.
J Diabetes Complications ; 34(8): 107613, 2020 08.
Article in English | MEDLINE | ID: mdl-32505477

ABSTRACT

Diabetes is a chronic disease, characterized by hyperglycemia, which refers to the elevated levels of glucose in the blood, due to the inability of the body to produce or use insulin effectively. Chronic hyperglycemia levels lead to macrovascular and microvascular complications. The macrovascular complications consist of peripheral artery disease (PAD), cardiovascular diseases (CVD) and cerebrovascular diseases, while the microvascular complications comprise of diabetic microangiopathy, diabetic nephropathy, diabetic retinopathy and diabetic neuropathy. Vascular endothelial dysfunction plays a crucial role in mediating both macrovascular and microvascular complications under hyperglycemic conditions. In diabetic microvasculature, the intracellular hyperglycemia causes damage to the vascular endothelium through - (i) activation of four biochemical pathways, namely the Polyol pathway, protein kinase C (PKC) pathway, advanced glycation end products (AGE) pathway and hexosamine pathway, all of which commutes glucose and its intermediates leading to overproduction of reactive oxygen species, (ii) dysregulation of growth factors and cytokines, (iii) epigenetic changes which concern the changes in DNA as a response to intracellular changes, and (iv) abnormalities in non-coding RNAs, specifically microRNAs. This review will focus on gaining an understanding of the molecular complexities underlying the vascular complications in diabetes mellitus, to increase our understanding towards the development of new mechanistic therapeutic strategies to prevent or treat diabetes-induced vascular complications.

18.
Apoptosis ; 25(5-6): 388-399, 2020 06.
Article in English | MEDLINE | ID: mdl-32418060

ABSTRACT

Type 2 diabetes has a strong association with the development of cardiovascular disease, which is grouped as diabetic heart disease (DHD). DHD is associated with the progressive loss of cardiovascular cells through the alteration of molecular signalling pathways associated with cell death. In this study, we sought to determine whether diabetes induces dysregulation of miR-532 and if this is associated with accentuated apoptosis. RT-PCR analysis showed a significant increase in miR-532 expression in the right atrial appendage tissue of type 2 diabetic patients undergoing coronary artery bypass graft surgery. This was associated with marked downregulation of its anti-apoptotic target protein apoptosis repressor with caspase recruitment domain (ARC) and increased TUNEL positive cardiomyocytes. Further analysis showed a positive correlation between apoptosis and miR-532 levels. Time-course experiments in a mouse model of type 2 diabetes showed that diabetes-induced activation of miR-532 occurs in the later stage of the disease. Importantly, the upregulation of miR-532 preceded the activation of pro-apoptotic caspase-3/7 activity. Finally, inhibition of miR-532 activity in high glucose cultured human cardiomyocytes prevented the downregulation of ARC and attenuated apoptotic cell death. Diabetes induced activation of miR-532 plays a critical role in accelerating cardiomyocytes apoptosis. Therefore, miR-532 may serve as a promising therapeutic agent to overcome the diabetes-induced loss of cardiomyocytes.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 2/genetics , MicroRNAs/genetics , Muscle Proteins/genetics , Aged , Aged, 80 and over , Animals , Antagomirs/genetics , Antagomirs/metabolism , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Cell Line , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Female , Gene Expression Regulation , Glucose/pharmacology , Glycated Hemoglobin A/genetics , Glycated Hemoglobin A/metabolism , Heart Atria/drug effects , Heart Atria/metabolism , Heart Atria/pathology , Humans , Male , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Middle Aged , Muscle Proteins/metabolism , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Signal Transduction , Triglycerides/blood
19.
Proc Natl Acad Sci U S A ; 117(1): 717-726, 2020 01 07.
Article in English | MEDLINE | ID: mdl-31871197

ABSTRACT

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.


Subject(s)
Asparagine/metabolism , Epithelial Sodium Channels/metabolism , Extracellular Matrix/metabolism , Protein Domains/genetics , Animals , Asparagine/chemistry , Disease Models, Animal , Endothelial Cells , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Epithelial Sodium Channels/chemistry , Epithelial Sodium Channels/genetics , Female , Glycosylation , HEK293 Cells , Humans , Hypertension/etiology , Hypertension/pathology , Hypertension/physiopathology , Male , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Oocytes , Patch-Clamp Techniques , Point Mutation , Polysaccharides/chemistry , Stress, Mechanical , Xenopus laevis
20.
Methods Mol Biol ; 2029: 161-173, 2019.
Article in English | MEDLINE | ID: mdl-31273741

ABSTRACT

Cardiac progenitor cells (CPCs) are gaining interest as a therapeutic option for the treatment of the heart. Due to the limited pool of CPCs residing in the heart, it is essential to isolate and expand the CPCs in vitro. Here we describe the protocol for isolation and culture of the heterogeneous population of CPCs from right atrial appendage and left ventricular tissue collected from patients undergoing on-pump coronary artery bypass graft surgery for the treatment of ischemic heart disease. Our protocol is developed to simultaneously isolate, culture, and characterize the CPCs from both atrial and ventricular tissues. We also describe the protocol for flow cytometry and immunohistochemical characterization of the isolated CPCs.


Subject(s)
Myocytes, Cardiac/cytology , Cells, Cultured , Coronary Artery Bypass/methods , Coronary Vessels/cytology , Flow Cytometry/methods , Heart Ventricles/cytology , Humans , Myocardium/cytology
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