Endothelial Adhesion Research Articles

P302 ENDOTHELIALITIS AS A POSSIBLE CAUSE OF MYOCARDIAL INJURY DURING COVID–19 INFECTION

A 53–year–old woman was admitted for chest pain for over two weeks, fever, dry cough, fatigue and myalgia. Chest pain increased in supine position and inspiration. Nasopharyngeal swab was positive for SARS–CoV–2 RT–PCR assay. Admission ECG displayed mild ST–segment elevation (0,5 mm) in inferolateral leads and echocardiogram showed normal left ventricular volume, mildly decreased left ventricular EF (52%) and no regional wall motion abnormalities. Blood tests exhibited elevated values of high–sensitivity troponin I (818 pg/mL), proBNP (19.190 pg/mL), CRP (42 mg/dL), D–Dimer test (1090 ng/mL) and WBC (13000/uL, N 89%, L 7%). IL–6 resulted slightly increased (18 ng/mL). ECG after 24 hours showed T wave inversion in inferolateral leads. Coronary CT angiography revealed normal epicardial arteries. A new echocardiogram, after five days, showed infero–basal hypokinesia, improved global EF (57%) and minimal pericardial effusion. Aspirin and fondaparinoux were started. Myopericarditis was suspected and high dose steroids were administered. After a few days, symptoms improved and ECG, echocardiographic abnormalities and troponin levels were normalized. At 6 weeks follow–up the patient was asymptomatic, ECG and echocardiogram were both normal. Instead cardiac MRI showed signs of endothelial injury: presence of interstitial oedema in the inferoapical and anteroseptal wall (STIR–T2–weighted), without late gadolinium enhancement. We measured blood levels of endothelial dysfunction markers: soluble ICAM–1, VCAM–1 and von Willebrand factor (vWF). They all resulted elevated as compared to a laboratory pool of normal serum (535 ng/ml, 265 ng/ml, and 1659 ng/ml, respectively). Our case was suggestive of myopericarditis related to Sars–Cov–2, but echocardiogram and MRI did not show signs of myocarditis permanent damage. However MRI showed interstitial oedema and hyperemia, typical signs of endothelial injury. Moreover we found high levels of soluble endothelial adhesion molecules, vWF, ICAM–1 and VCAM–1, likely expression of the microcirculatory endothelial dysfunction due to myocardial endothelialitis.In conclusion, our case report suggests that, in COVID–19, acute myocardial injury associated with clinical and investigational signs of myocarditis can be due to myocardial endothelialitis rather than true myocarditis and that an early management with anti–inflammatory drugs (steroids) can be particularly beneficial in patients with unexplained elevated cardiac troponin.

GSTM1 Copy Number and Kidney Disease in People With HIV

GSTM1 Copy Number and Kidney Disease in People With HIV

Potential Mechanisms and Effects of Chinese Medicines in Treatment of Diabetic Atherosclerosis by Modulating NLRP3 Inflammasome: A Narrative Review.

Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) is an intracellular sensor that detects endogenous danger signals and environmental irritants to assemble into the NLRP3 inflammasome. Activation of the NLRP3 inflammasome leads to the secretion of the proinflammatory cytokines interleutkin (IL)-1β and IL-18 and induces pyroptosis. Recent studies have shown that the NLRP3 inflammasome participates in the initiation and progression of diabetic atherosclerosis through pathological mechanisms such as β-cell dysfunction, insulin resistance, endothelial cell dysfunction, monocyte adhesion and infiltration, and smooth muscle cell proliferation and migration. In diabetic atherosclerosis, Chinese medicine has been proven effective for the inflammatory response mediated by the NLRP3 inflammasome. This review summarizes the latest progress on the NLRP3 inflammasome in the pathogenesis and potential Chinese medicine treatment of diabetic atherosclerosis.

Neutrophil Protein Kinase R Mediates Endothelial Adhesion and Migration by the Promotion of Neutrophil Actin Polymerization.

Neutrophils protect against bacterial and fungal infections, but tight regulation of cell activation is essential for avoiding tissue damage in autoimmune disorders. Protein kinase R (PKR) is a serine/threonine kinase originally characterized by its role in the defense mechanisms against viral infection. Although PKR is involved in the signaling pathways of neurodegenerative diseases and metabolic disorders, its function in neutrophils is not well delineated. In this study, we demonstrate that human neutrophil PKR mediates adhesion to endothelial cells under physiological flow conditions but does not mediate rolling on those cells. Also, neutrophil PKR activation contributes to migration toward chemoattractants. Mechanistically, neutrophil PKR mediates the cell spreading and binding to ICAM-1 in static condition. Moreover, Ab microarray reveals that calcium/calmodulin-dependent protein kinase II is phosphorylated downstream of PKR and affects actin polymerization that is a cytoskeleton rearrangement indispensable for neutrophil migration induced by fMLF. In vivo, neutrophil recruitment into the dorsal air pouch of mice is reduced by PKR inhibitor treatment. Also, in mice with nephrotoxic serum nephritis, the compound treatment suppresses neutrophil accumulation in kidney glomerulus and subsequent development of albuminuria. Thus, in vascular inflammation, neutrophil PKR plays a critical role in the recruitment process, including endothelial adhesion and migration via leukocyte actin polymerization.

Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks.

BackgroundThe pathophysiology of diabetic retinopathy (DR) is multifaced. A low level of circulating adiponectin (APN) in type 2 diabetes is associated with microvasculature complications, and its role in the evolution of DR is complex.AimThis study is designed to explore the potential impact of APN in the pathogenesis of DR, linking the changes in cellular and biological processes with the pathways, networks, and regulators involved in its actions.MethodsHuman microvascular retinal endothelial cells (HMRECs) were exposed to 30mM glucose (HG) and treated with globular adiponectin (30μg/mL) for 24 hours. The cells were evaluated for reactive oxidative stress (ROS) and apoptosis. RT-PCR profile arrays were utilized to evaluate the profile of genes involved in endothelial functions, angiogenesis, extracellular matrix, and adhesion molecules for hyperglycemic HMRECs treated with adiponectin. In addition, the barrier function, leukocyte migration, and angiogenesis were evaluated. The differential expressed genes (DEGs) were outlined, and bioinformatic analysis was applied.ResultsAdiponectin suppresses ROS production and apoptosis in HMRECs under HG conditions. Adiponectin improved migration and barrier functions in hyperglycemic cells. The bioinformatic analysis highlighted that the signaling pathways of integrin, HMGB1, and p38 AMPK, are mainly involved in the actions of APN on HMRECs. APN significantly affects molecular functions, including the adhesion of cells, chemotaxis, migration of WBCs, and angiogenesis. STAT3, NFKB, IKBKB, and mir-8 are the top upstream regulators, which affect the expressions of the genes of the data set, while TNF and TGFB1 are the top regulators.ConclusionAdiponectin significantly counteracts hyperglycemia at various cellular and molecular levels, reducing its impact on the pathophysiological progression towards DR in vitro using HMRECs. Adiponectin ameliorates inflammatory response, oxidative stress, and endothelial barrier dysfunction using a causal network of NFBk complex, TNF, and HMGB1 and integrin pathways.

Biophysical Properties of Endothelial Mechanotransduction Mediated by Glycocalyx

Endothelial surface glycocalyx (ESG) is a carbohydrate‐rich layer found on the endothelial cell (EC) surface lining all blood vessels' inner lumen. Composed of membrane glycoproteins, glycosaminoglycans (GAGs), such as hyaluronan and heparan sulfate (HS), and proteoglycans, the ESG forms a bulky, gel‐like layer serving critical functions in blood flow mechanotransduction, endothelial permeability maintenance, leukocyte adhesion, and inflammation control. ESG is known for sensing shear stress of blood flow and transducing the mechanical signal into nitric oxide (NO) production. NO is an essential signaling molecule to regulate vascular tone. To date, the molecular pathways of ESG‐mediated mechanotransduction have not been completely clear.We utilize a custom‐built AFM with fluorescence imaging capabilities to vertically stretch the ESG, which applies a pulling force to specific ESG components on the surface of a single live mouse brain endothelial cell (bEnd.3) and quantify the cell's NO production in real‐time. NO production is quantified by the fluorescent dye DAF‐FM. AFM probe is coated with monoclonal antibodies against HS or glypican‐1 (a major core protein for HS) to exert pulling forces onto the HS or glypican‐1 on bEnd.3. To ensure that DAF intensity is a signal of NO production, we treated the cells with L‐NAME (an inhibitor of endothelial nitric oxide synthase). The DAF intensity was greatly reduced, which confirms that the DAF intensity is representing NO production.The AFM anti‐glypican‐1 coated probe showed an increase in DAF intensity, while the anti‐HS probe is shown to have a slight rise in DAF intensity. Since HS may attach to other proteins besides glypican, the data suggest that glypican‐1 is a more critical mechanotransducer. Our prior study indicated cation‐permeable, transient receptor potential (TRP) ion channels mediate stretch‐induced NO production. Probes were coated in glypican‐1 while the cells were exposed to Amiloride or SKF96365, blockers of TRPP2 and TRPC1 channels, respectively. After the Amiloride‐treated cells were mechanically stimulated, there was a reduction of NO production. However, the amount of NO output was still significant over the basal levels observed in cells. When the SKF96365‐treated cells were mechanically stimulated, production was inhibited entirely, thus, demonstrating TRPC1 channels play a vital role in allowing the Ca2+ into the cell to begin NO production cascade.These findings confirm the critical roles of HS, glypican‐1, and TRP channels on the rapid NO production resulting from mechanical signaling and have important implications for ESG‐related cardiovascular diseases such as diabetes, strokes, cardiac arrest, and atherosclerosis. It has been shown that intracellular Ca2+regulates eNOS activity. In future studies, we will measure the Ca2+intake mediated by TRP channels and delineate the interplay among mechanical stretch, TRP channel activation, Ca2+intake and NO production.

Mice Deficient in Endothelial Cell‐Selective Adhesion Molecule Develop Vascular Endothelial and Left Ventricle Diastolic Dysfunction

The Endothelial Cell‐Selective Adhesion Molecule (ESAM) mediates inflammatory cell transmigration and also involved in pathological angiogenesis. Here we investigated the role of ESAM in affecting vasodilator and cardiac contractile function. To that end ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed left ventricle (LV) diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail‐cuff plethysmography, was not different between ESAM knockout and WT mice, whereas the heart to body weight ratio is increased in ESAM knockout mice. Using an unbiased automated tracing of the microvasculature we found a decreased vascularization (total vascular length) of the myocardium in ESAM knockout mice. In addition, vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium‐dependent, acetylcholine‐induced relaxation of the pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium‐independent, sodium nitroprusside‐induced relaxation was similar in two groups. Thus, we conclude that ESAM deficiency causes vasodilator dysfunction, impaired angiogenesis underlying the development LV diastolic dysfunction.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Abstract Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. To dissect early lymphoid specification events, we generated two transgenic mouse models reporting and tracing the expression of terminal deoxynucleotidyl transferase (TdT). Surprisingly, transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset which lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction by MPPs reflected a transcriptionally dynamic but uncommitted stage characterized by low expression of lineage-associated genes. Further, analysis using single-cell CITE-Seq indicated that multipotency is associated with expression of Endothelial Cell-Selective Adhesion Molecule (ESAM). Developmental progression along all lineages is defined by downregulation of ESAM and by stable TdT expression within the lymphoid trajectory. Collectively, we propose a new developmental hierarchy of early hematopoietic progenitors for which we provide their transcriptional profile as well as their phenotypic and functional properties at single cell resolution. Supported by NIH intramural funding

IL‐33/ST2L Signaling Regulate Blood‐Retinal Barrier Integrity via Affecting Cadherin‐Catenin Adheren Junction Complex

PurposeAngiogenesis, neovascularization and vascular remodeling are highly dynamic processes, where endothelial cell‐cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity and barrier function. The cadherin–catenin adhesion complex is a key contributor to blood‐retinal barrier (BRB) integrity and dynamic cell movements. However, the pre‐eminent role of cadherins and their associated catenins in adheren junction structure and function is not fully understood. Also, the role of IL33/ST2L signaling in regulating BRB integrity is not known.MethodsUsing a murine model of oxygen‐induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we are trying to understand the significance of IL‐33/ST2L‐signaling on endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability.ResultsOur in vivo genetic deletion studies revealed that deletion of IL‐33 resulted in reduced vascular tufts and vascular leakage. Therefore, in order to understand the molecular mechanism by which IL‐33 regulates the BRB integrity, we performed some in vitro studies using HRMVECs. The electric cell‐substrate impedance sensing (ECIS) analysis showed that IL‐33 (20 ng/ml) induce endothelial barrier disruption in HRMVECs. Likewise, FITC‐dextran permeability assay also showed that IL‐33 stimulate the endothelial dysfunction in HRMVECs. As we know, tight junctions (TJs) and adheren junctions (AJs) form the adhesion structure and maintain the BRB integrity, therefore we look for the junctional proteins involved in the IL‐33/ST2L mediated endothelial dysfunction. Furthermore, we found that IL‐33 mediates the phosphorylation of VE‐cadherin and a‐catenin in HRMVECs. Furthermore, mass‐spectroscopy (MS) analysis revealed the phosphorylation of specific Ser439 residue of a‐catenin upon IL‐33 inducement in HRMVECs. Consequently, mutation of a‐catenin at Ser439 residue restores the BRB integrity disrupted by IL‐33. In addition, we have observed that IL‐33 induce the generation of reactive oxygen species (ROS), thereby destabilize adheren junctions in HRMVECs.ConclusionsWe conclude that IL‐33/ST2L signaling plays a significant role in angiogenesis and neovascularization by regulating endothelial permeability and BBB integrity.

Pathophysiology of Venous Thromboembolism: Putative role of HDL

IntroductionIn 2019, cardiovascular disease (CVD) was the main reason of all global deaths (32%) and out of these deaths, 85% were due to heart attack and stroke alone. Venous thromboembolism (VTE), which is the third leading cause of CVD, has a very high incidence rate in Indian Population. Data suggest that thromboembolic disorders are due to multiple genetic and environmental variables and hence considered as a complex multifactorial trait. High Density Lipoprotein (HDL) has attracted a great deal of interest in recent years because of its implications in the prevention of various CVD. Various data suggests the relation between HDL and the expression of coagulation/fibrinolytic factors among the patients of CVD. Protein content of HDL is largely responsible for antioxidant mechanism by metabolizing lipid hydro peroxides and preventing their accumulation. Association of venous thrombosis and dyslipidemia has been investigated in several clinical studies, but results are inconsistent. Therefore, we sought to identify the role of HDL in pathophysiology of VTE in both human and animal studies.MethodologyDVT was detected in male patients using color Doppler followed by D‐Dimer and Prothrombin Fragment 1+2 (PF1+2) levels to confirm thrombus formation. HDL associated proteins, endothelial adhesion molecules, thrombotic molecules, inflammatory molecules and oxidized lipoprotein levels were measured. For animal study, a complete stasis animal model was standardized to generate thrombus formation in the SD rats. Thrombus length/weight was measured along with D‐Dimer & PF 1+2 levels. HDL was isolated from plasma and was used in kinetic reaction of LDL oxidation. Progression of LDL oxidation was measured through DCFH‐method. All experimental procedures were performed in accordance to ethical guidelines.Major FindingsA well written consent was taken from all the participants before conducting human study. High levels of D‐Dimer and PF 1+2 in DVT patients as well as IVC ligated group confirmed thrombosis. HDL associated molecules were found to be significantly lower, whereas adhesion molecules, inflammatory molecules, and thrombotic molecules were significantly higher in both DVT patients and IVC ligated group. Oxidized lipoproteins and the lipooxygenase enzymes responsible for lipoprotein oxidation were found significantly higher. Isolated HDL used in LDL oxidation kinetics reaction found to be non‐functional in preventing LDL oxidation. P‐value of <0.05 was used as significance for all the tests.Conclusion & DiscussionLow levels of HDL proteins along with high levels of adhesion molecules, thrombotic markers and inflammatory molecules indicate the suppression of HDL activity. High lipooxygenase enzymes and oxidized lipoprotein levels support the hypothesis of dysfunctional HDL. HDL isolated from the ligated group was unable to prevent the LDL oxidation which clarifies that HDL loses its functionality in case of VTE. Furthermore, finding of this study will also help in understanding the pathophysiology of VTE which might give an insight in prevention/early diagnosis of the disease.

ITGA4 knockout prevents blood-brain-barrier migration of chimeric antigen receptor T cells

Abstract Chimeric Antigen Receptor (CAR) T cells have shown remarkable anti-tumor activity in various B cell malignancies. However, expanding their use to solid tumors has proven difficult due to the lack of sufficiently tumor-specific target antigens. Restricting CAR T cells from trafficking to certain healthy tissues showing shared expression of an otherwise tumor-specific target antigen may open up new avenues to the treatment of solid tumors with CAR T cells. We developed an electroporation-based CRISPR/Cas9 approach targeting integrin a4 (ITGA4), a key component of the T cell adhesion heterodimer VLA-4 required for blood-brain-barrier migration via the endothelial adhesion protein VCAM-1. We characterize ITGA4 knockout kinetics in human T cells by flow cytometry, determine its effect on T cell adhesion to VCAM-1 using a custom in vitro adhesion assay, determine tissue distribution of ITGA4ko cells in a mouse model and characterize the anti-tumor activity of ITGA4ko CAR T cells using a luminescence-based cytotoxicity assay. Near-complete ITGA4 knockout could be achieved during a standard 12-day CAR T cell production process and significantly decreased adhesion of human T cells to VCAM-1. ITGA4ko T cells appeared to be efficiently redirected from the brain to the bone marrow which was validated by flow cytometry showing almost a complete lack of T cells in the brain after ITGA4 knockout. Importantly, knockout did not significantly change CAR T cell expansion or anti-tumor activity. In conclusion, we show that ITGA4 knockout is feasible, results in efficient exclusion of T cells from the brain without affecting CAR T cell function, representing a promising approach to shape CAR T cell selectivity for the treatment of solid tumors.

PO-25: Elevated plasma von Willebrand Factor in patients with breast cancer may contribute to the early metastatic niche, promoting endothelial adhesion, invasion and angiogenic properties of circulating breast cancer cells

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Dehydroabietic Acid Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice and VCAM-1 Expressions in-vitro

Dehydroabietic acid (DHA) is an analog of abietic acid (AA) for cardiovascular disease prevention, known to act pharmacologically against aging, inflammation, bacterial infections and cancer. The current research investigated the molecular mechanisms of DHA on the adhesiveness of endothelial leukocytes and activation of NF-κB in TNF-α treated HAEC and ApoE−/− mice in experimental atherosclerosis. The HAECs were tested for toxicity using MTT assay at various DHA concentrations. The cell-surface expression of CAM against endothelial leukocyte adhesion molecule-1, ICAM -1, or human VCAM-1 was determined by the ELISA test, followed by western blot analysis. Endothelial cell-leukocyte adhesion assay involving U937 cells was carried out followed by the determination of NF-κB p65 expression. ApoE−/− mice were fed with a high cholesterol diet every day followed by oral administration of DHA (10 and 20 mg/kg/day). The DHA (5 and 10 mM) substantially reduced (p < 0.05) human (U937) cell lines binding to TNF-α activated human endothelial aortic cells. The assays involving the 32P-labelled NF-kB as a probe demonstrated that DHA pre-treatment decreased the shifted bands density following the stimulation of TNF-α. Nuclear extracts immune blot assessment and immune fluorescence staining showed a significant decrease (p < 0.05) in the NF-κB p65 concentration in the nuclei with DHA treated human endothelial aortic cells. Together these findings indicate that DHA inhibits nuclear trans localization of TNF-α-induced NF-κB p65, and thus considerably suppresses (p < 0.05) the VCAM-1 expression, which contributes to lower leukocyte adherence suggesting that DHA helps in preventing inflammatory atherosclerosis in vivo.

Endothelial miR-199a-3p regulating cell adhesion molecules by targeting mTOR signaling during inflammation

BackgroundAdherence of monocytes to endothelial cells is the initial stage for development of coronary artery disease (CAD). MiRNAs have been reported to participate in this process by regulating the expression of cell adhesion molecules. This study aimed to explore the function of miR-199a-3p in endothelial inflammation and adhesion. MethodsWe assessed the expression of miR-199a-3p in CAD patients and ApoE-/- mice. The relationship between miR-199a-3p level and endothelial inflammation and adhesion was examined. ELISA was used to test the level of IL-6 and IL-8. Dual luciferase reporter assay was used to evaluate the binding between miR-199a-3p and mTOR. ResultsA decreased expression of miR-199a-3p was observed in the PBMCs and plasma of CAD patients, aorta of ApoE-/- mice and inflammatory HUVECs. MiR-199a-3p significantly suppressed the expression levels of pro-inflammatory cytokine (IL-6, IL-8), endothelial adhesion molecules (ICAM-1, VCAM-1) and monocyte-endothelial cells interaction. MiR-199a-3p directly targeted and repressed mTOR, and its suppression effect on ICAM-1 and VCAM-1 was abolished by mTOR inhibitor rapamycin, and rescued by mTOR activator MHY1485. Overexpression of miR-199a-3p promoted autophagy in HUVECs and inhibiting autophagy by chloroquine attenuated the effect of miR-199a-3p on ICAM-1 and VCAM-1 expression. Inhibition of autophagy promoted endothelial adhesion molecule expression and monocyte-EC interaction. ConclusionsOur results suggested that miR-199a-3p suppressed endothelial inflammation and adhesion by targeting mTOR signaling and increasing autophagy. Our findings point to an important role for miR-199a-3p in the early stage of cardiovascular disease.

Impact of Anti-Endothelial Cell Antibodies (AECAs) in Patients with Polycythemia Vera and Thrombosis.

Polycythemia vera (PV) causes thrombosis. Erythrocytosis and cell adhesiveness are responsible for thrombosis. JAK2V617F causes inflammation and autoimmunity; however, whether or not autoimmunity or inflammation causes thrombosis has yet to be proven. In 60 PV patients, we analyzed JAK2V671F and its allele burden, autoimmune Th17 cells, interleukin-17 (IL-17), anti-endothelial cell antibodies (AECAs), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor antigen (VWF:Ag). Fifty blood donors were used as the controls. All patients were on phlebotomy-maintaining hematocrit <45% and aspirin. Of the 60 patients, 40 had thrombosis. Those patients with thrombosis had a higher JAK2V617F allele burden than those without thrombosis, andTh17 cells and IL-17 were also higher in patients with thrombosis. Interestingly, we observed a high AECA IgG ELISA ratio (ER) in patients with thrombosis, which was normal in patients without thrombosis. We found high ELAM-1 and ICAM-1 as well as high VWF:Ag in patients with thrombosis compared to patients without thrombosis. AECA-positive sera from patients with thrombosis showed enhanced binding to cytokine-treated HUVEC and a positive antibody-dependent cellular cytotoxicity, suggesting that AECA may contribute to vascular injury. A positive correlation between AECAs, allele burden, and thrombosis was found. These results suggest that autoimmunity may be an additional mechanism in PV thrombogenesis.

Sirt7 associates with ELK1 to participate in hyperglycemia memory and diabetic nephropathy via modulation of DAPK3 expression and endothelial inflammation

Diabetic nephropathy (DN) is one of the most serious complications of advanced diabetes, and increases patient mortality. Recently, epigenetics-mediated hyperglycemic memory in pathological process of DN has received attention. The purpose of this study was to determine the underlying mechanism by which sirt7 modulates hyperglycemic memory in DN. In glomerular endothelial cells (GECs) cultured in high glucose and glomeruli of DN patients and rats, an increase in p65 phosphorylation and endothelial adhesion molecule levels persisted after glucose normalization but was reversed by glucose normalization associated with death-associated protein kinase-3 (DAPK3) knockout or DAPK3 inhibitor. High glucose-mediated decrease in sirt7, the deacetylase modulating H3K18-acetylation (H3K18ac), was sustained after normoglycemia. Sirt7 overexpression accompanied by glucose normalization suppressed DAPK3 expression and inflammation in GECs. Moreover, sh-sirt7-induced inflammation was inhibited by si-DAPK3. Furthermore, sirt7 and H3K18ac were located at the DAPK3 promoter region. ELK1 was found to combine with sirt7. si-ELK1 supplemented with normoglycemia inhibited high glucose-induced DAPK3 expression and inflammation in GECs. ELK1 overexpression-mediated inflammation was inhibited by si-DAPK3. In addition, ELK1 and sirt7 were located at the same promoter region of DAPK3. ELK1 overexpression enhanced DAPK3 promoter activity, which disappeared after specific binding site mutation. In vivo, sirt7 overexpression decreased inflammation and improved renal function during insulin treatment of DN rats, whereas insulin alone did not work. Our data demonstrated high glucose-mediated mutual inhibition between sirt7 and ELK1 induced DAPK3 transcription and inflammation despite normoglycemia in GECs, thus forming a vicious cycle and participating in the occurrence of hyperglycemic memory in DN.

3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM).

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Quercetin Mitigates Endothelial Activation in a Novel Intestinal-Endothelial-Monocyte/Macrophage Coculture Setup.

Atherosclerosis initiation is associated with a pro-inflammatory state of the endothelium. Quercetin is a flavonoid abundantly present in plant-based foods, with a possible impact on cardiovascular health. In this study, the effects of quercetin on lipopolysaccharide (LPS)-mediated endothelial inflammation and monocyte adhesion and migration, which are initial steps of the atherogenic process, are studied. Novel in vitro multicellular models simulating the intestinal-endothelial-monocytes/macrophages axis allowed to combine relevant intestinal flavonoid absorption, metabolism and efflux, and the consequent bioactivity towards peripheral endothelial cells. In this triple coculture, quercetin exposure decreased monocyte adhesion to and macrophage migration through an LPS-stressed endothelium, and this was associated with significantly lower levels of soluble vascular cell adhesion molecule-1 (sVCAM-1). Furthermore, quercetin decreased the pro-inflammatory cell environment upon LPS-induced endothelial activation, in terms of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and sVCAM-1 expression. These findings highlight a mode-of-action by which quercetin may positively impact the initial states of atherosclerosis under more physiologically relevant conditions in terms of quercetin concentrations, metabolites, and intercellular crosstalk.

Influence of a proinflammatory state on postprandial outcomes in elderly subjects with a risk phenotype for cardiometabolic diseases

PurposeLow-grade inflammation in obesity is associated with insulin resistance and other metabolic disturbances. In response to high-energy meal intake, blood concentrations of inflammatory markers, glucose and insulin rise. The aim of this study was to examine whether a basal inflammatory state influences postprandial responses.MethodsA randomized crossover trial was performed in 60 participants with a cardiometabolic risk phenotype (age 70 ± 5 years; BMI 30.9 ± 3.1 kg/m2). Each participant consumed three different iso-energetic meals (4300 kJ): a Western diet-like high-fat meal (WDHF), a Western diet-like high-carbohydrate meal (WDHC) and a Mediterranean diet-like meal (MED). Blood samples were collected when fasted and hourly for 5 h postprandially and analyzed for glucose, insulin, interleukin-1β (IL-1β), interleukin-6 (IL-6) and endothelial adhesion molecules. Based on fasting serum C-reactive protein (CRP) concentrations, participants were assigned to a high inflammation (CRP ≥ 2.0 mg/L; n = 30) or low inflammation (CRP < 2.0 mg/L; n = 30) group, and postprandial outcomes were compared.ResultsPlasma IL-6, glucose and serum insulin increased after all meals, while IL-1β and endothelial adhesion molecules were unchanged. The high inflammation group had higher fasting and postprandial IL-6 concentrations than the low inflammation group, although the IL-6 response slope was similar between groups. In response to the WDHC meal, participants in the high inflammation group experienced a higher glycaemic response than those in the low inflammation group.ConclusionA basal proinflammatory state results in higher absolute fasting and postprandial IL-6 concentrations, but the increase in IL-6 relative to basal levels is not different between high and low inflammation groups. Elevated glycaemic response in the high inflammation group may be due to inflammation-induced short-term insulin resistance.The trial was registered at http://www.germanctr.de and http://www.drks.de under identifier DRKS00009861 (registration date, January 22, 2016).

Serum miR-497-5p serves as a diagnostic biomarker for acute coronary syndrome and predicts the occurrence of major adverse cardiovascular events after percutaneous coronary intervention

ABSTRACT This study aimed to investigate the diagnostic value of microRNA (miR)-497-5p in acute coronary syndrome (ACS) and its predictive value for the occurrence of adverse major adverse cardiovascular events (MACEs). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of serum miR-497-5p in 110 ACS patients and 82 controls. And miR-497-5p levels were found to be significantly elevated in the patients (P < 0.001). Pearson correlation coefficient confirmed that miR-497-5p was positively correlated with Gensini scores (r = 0.684). The area under the Receiver-operating characteristic (ROC) curve was 0.861, which significantly identified patients with ACS, and was confirmed by logistic regression (OR = 8.533, 95%CI = 4.113–17.787, P < 0.001). Kaplan-Meier and Cox regression was performed to evaluate the predictive value of miR-497-5p in the occurrence of MACEs during a 6-month follow-up after percutaneous coronary intervention (PCI) in patients with ACS. The results demonstrated that miR-497-5p was an independent predictor of MACEs (HR = 4.773, 95%CI = 1.569–12.036, P = 0.013) and that patients with high level of miR-497-5p were more likely to develop MACEs after PCI (long-rank P = 0.019). Finally, miR-497-5p positively correlated with endothelial proinflammatory and adhesion factors. Our study suggests that serum miR-497-5p is a potential diagnostic marker for ACS and its elevated levels can predict a high risk of MACEs in ACS patients after PCI. And this may be associated with vascular endothelial injury.