Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

A Link Between Methylglyoxal and Heart Failure During HIV-1 Infection

Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.

Correlation Between Ultrawide-Field Fluorescence Contrast Results and White Blood Cell Indexes in Diabetic Retinopathy

Background: Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. DR involves a state of systemic inflammation, and chronic inflammation can promote microvascular and macrovascular diseases in diabetic patients and accelerate disease progression. Ultrawide-field FFA (UWFA) systems are increasingly being used to examine a wider retina. To explore the correlation between the different manifestations of retinopathy under UWFA and the systemic indicators of white blood cells in patients with diabetic retinopathy .Methods: This retrospective study included the hospitalized DR patients in the Department of Ophthalmology and Endocrinology of the Affiliated Hospital 2 of Nantong University between January 2016 and March 2019. This study examined the correlations between the UWFA examination results and glycated hemoglobin (HbA1c), routine blood tests, blood coagulation function, liver and kidney function, and the neutrophil-to-lymphocyte ratio of patients with clinically diagnosed DR during hospitalization.Results: A total of 115 patients with DR (53 females and 62 males) were included (199 eyes: 102 right eyes and 97 left eyes). UWFA revealed that most eyes (77.4%) had grade 4 microvascular leakage, 52.8% had grade 0 capillary non-perfusion area, 59.3% had grade 0 neovascularization, and 92.0% had grade 0 fibrous proliferative membranes. Microvascular leakage was correlated with the NLR (r=0.186, P=0.027). Capillary non-perfusion area was correlated with the monocyte ratio (r=0.144, P=0.042) and the eosinophil ratio (r=0.123, P=0.044). Neovascularization was correlated to the monocyte ratio (r=0.324, P=0.018). Finally, the fibrous proliferative membrane was correlated to the monocyte ratio (r=0.418, P=0.002). Only the eosinophil ratio was independently associated with proliferative DR (odds ratio=1.25, 95% confidence interval: 1.04-1.51, P=0.018).Conclusion: The results of UWFA imaging in patients with DR are correlated with white blood cell population indexes. The eosinophil ratio was independently associated with proliferative DR.

Influenza A virus infects pulmonary microvascular endothelial cells leading to microvascular leakage and release of pro-inflammatory cytokines

Objective To investigate the replication of influenza A virus A/Puerto Rico/8/34 (H1N1) in pulmonary microvascular endothelial cells and its effect on endothelial barrier function. Methods Human pulmonary microvascular endothelial cells were infected with influenza A/Puerto Rico/8/34 (H1N1) virus. Plaque reduction assay, real-time quantitative PCR, immunofluorescence staining, and western blot were used to elucidate the replication process of virus-infected endothelial cells. In addition, real-time quantitative PCR was used to detect the relative expression levels of mRNA of some inflammatory factors. The endothelial resistance assay was used to determine the permeability of the endothelial monolayer. Excavation and analysis of data from open databases, such as the GeneCards database, DAVID Bioinformatics Resources, STRING search tool, and DGIdb database determined the genes, proteins, and signal pathways related to microvascular leakage caused by the H1N1 virus, and predicted the drugs that could be effective for treatment. Results In vitro experiments showed that the influenza virus can infect endothelial cells, leading to a significant increase in the permeability of pulmonary microvascular endothelial cells and the release of pro-inflammatory cytokines, but does not efficiently replicate in endothelial cells. A total of 107 disease-related target genes were obtained from the Gene-cards database. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes mainly affected the pathways related to “Inflammatory bowel disease” (IBD), “Chagas disease” (American trypanosomiasis), “Influenza A”, and also played a key role in anti-inflammation and regulation of immunity. After enrichment analysis, 46 hub genes were screened. A total of 42 FDA-approved drugs corresponding to the hub genes were screened from the DGIdb database, and these could be formulated for topical application. In addition, these drugs can be used to treat other diseases, including cancer, inflammatory diseases, immune system disorders, and cardiovascular diseases. Conclusion H1N1 influenza virus affects the barrier function of endothelial cells indirectly. Combined with bioinformatics tools, we can better understand the possible mechanism of action of influenza A (H1N1) virus causing pulmonary microvascular leakage and provide new clues for the treatment of pulmonary microvascular leakage.

Characterization of Urothelial Inclusions in Male Wistar Han Rats Treated Orally With the Novel α2A-Adrenoceptor Agonist Tasipimidine

Intracellular inclusions were observed in urinary bladder epithelium of male Wistar rats, following oral treatment with high doses of the α2A-adrenoceptor agonist tasipimidine for 28 days. No cell death or inflammation was associated with the brightly eosinophilic inclusions. Electron microscopy (EM) studies showed that the inclusions represented intact or fragmented red blood cells (RBC) resulting from erythrophagocytosis, further supported by the presence of iron in urothelial cells. In addition, scattered iron-positive macrophages were observed in the submucosa and muscle layer, indicating microvascular leakage, as no major hemorrhage was evident. Despite the presence of inclusions, the urothelium showed normal uroplakin III distribution, normal cell turnover, and an absence of α-2u-globulin. It is, therefore, concluded that the inclusions were not associated with urothelial damage or increased renewal of the epithelium. This finding shows also that urothelial cells have the capability to phagocytize and break down RBCs originating from submucosal microvascular leakage. Similar changes were not observed in tasipimidine-treated beagle dogs (28 days), suggesting these findings were rat specific. The leakage of RBCs into the urothelium is suggested to be a consequence of exaggerated pharmacology leading to vasoconstriction of submucosal blood vessels in combination with transient increased bladder distension and pressure.

Adeno-Associated Viral Transfer of Glyoxalase-1 Blunts Carbonyl and Oxidative Stresses in Hearts of Type 1 Diabetic Rats

Accumulation of methylglyoxal (MG) arising from downregulation of its primary degrading enzyme glyoxalase-1 (Glo1) is an underlying cause of diabetic cardiomyopathy (DC). This study investigated if expressing Glo1 in rat hearts shortly after the onset of Type 1 diabetes mellitus (T1DM) would blunt the development of DC employing the streptozotocin-induced T1DM rat model, an adeno-associated virus containing Glo1 driven by the endothelin-1 promoter (AAV2/9-Endo-Glo1), echocardiography, video edge, confocal imaging, and biochemical/histopathological assays. After eight weeks of T1DM, rats developed DC characterized by a decreased E:A ratio, fractional shortening, and ejection fraction, and increased isovolumetric relaxation time, E: e’ ratio, and circumferential and longitudinal strains. Evoked Ca2+ transients and contractile kinetics were also impaired in ventricular myocytes. Hearts from eight weeks T1DM rats had lower Glo1 and GSH levels, elevated carbonyl/oxidative stress, microvascular leakage, inflammation, and fibrosis. A single injection of AAV2/9 Endo-Glo1 (1.7 × 1012 viron particles/kg) one week after onset of T1DM, potentiated GSH, and blunted MG accumulation, carbonyl/oxidative stress, microvascular leakage, inflammation, fibrosis, and impairments in cardiac and myocyte functions that develop after eight weeks of T1DM. These new data indicate that preventing Glo1 downregulation by administering AAV2/9-Endo-Glo1 to rats one week after the onset of T1DM, blunted the DC that develops after eight weeks of diabetes by attenuating carbonyl/oxidative stresses, microvascular leakage, inflammation, and fibrosis.