Beclin1 controls caspase-4 inflammsome activation and pyroptosis in mouse myocardial reperfusion-induced microvascular injury.

BACKGROUND: Myocardial reperfusion injury is often accompanied by cell death and inflammatory reactions. Recently, pyroptosis is gradually recognized as pivotal role in cardiovascular disease. However, little is known about the regulatory role of beclin1 in the control of caspase-4 activation and pyroptosis. The present study confirmed whether beclin1 regulates caspase-4 mediated pyroptosis and thereby protects Human Cardiac microvascular endothelial cells (HCMECs) against injury. METHODS: TTC and Evan's blue dye, western blot, immunofluorescence and immunohistochemistry staining were performed in wild mice and transgenic mice with overexpression of beclin 1(BECN1-Tg). CMECs were transfected with a beclin1 lentivirus. The cell cytotoxicity was analyzed by LDH-Cytotoxicity Assay Kit. The protein levels of autophagy protein (Beclin1, p62 and LC3II/LC3I) and caspase-4/GSDMD pathway were determined by western blot. Autophagic vacuoles in cells were monitored with RFP-GFP-LC3 using fluorescence microscope. RESULTS: I/R caused caspase-4 activity and gasdermin D expression increase in vivo and in vitro. Overexpression of beclin1 in heart tissue and CMECs suppressed the caspase-4 activity and decreased the levels of gasdermin D; meanwhile beclin1 overexpression also reduced IL-1ß levels, promoted autophagy (p62 expression was inhibited while LC3II expression was increased) in the heart and CMECs. Interestingly, beclin1 overexpression increased animal survival and attenuated myocardial infarct size (45 ± 6.13 vs 22 ± 4.37), no-reflow area (39 ± 5.22 vs 16 ± 2.54) post-myocardial ischemia reperfusion. CONCLUSIONS: Induction of beclin-1 signaling can be a potential therapeutic target in myocardial reperfusion-induced microvascular injury. Video Abstract.

CMR in the diagnosis of ischemic heart disease.

Cardiovascular magnetic resonance has always been more often used in the last 10 years in evaluation of heart disease. Role in diagnosis of ischemia and in evaluation of myocardial infarction is well established by many scientific papers and included in current guidelines. High accuracy in evaluation of stress-induced ischemia, tissue characterization and functional parameters are the pillars the make the method widely used. In this paper are described role and techniques in diagnosis of ischemia, myocardial infarction and its sequelae.

Sex-Dependent Molecular Mechanisms of Lipotoxic Injury in Brain Microvasculature: Implications for Dementia.

Cardiovascular risk factors and biologic sex play a role in vascular dementia which is characterized by progressive reduction in cognitive function and memory. Yet, we lack understanding about the role sex plays in the molecular mechanisms whereby lipid stress contributes to cognitive decline. Five-week-old low-density lipoprotein deficient (LDL-R -/-) male and female mice and C57BL/6J wild types (WT) were fed a control or Western Diet for 8 weeks. Differential expression of protein coding and non-protein coding genes (DEG) were determined in laser captured hippocampal microvessels using genome-wide microarray, followed by bioinformatic analysis of gene networks, pathways, transcription factors and sex/gender-based analysis (SGBA). Cognitive function was assessed by Y-maze. Bioinformatic analysis revealed more DEGs in females (2412) compared to males (1972). Hierarchical clusters revealed distinctly different sex-specific gene expression profiles irrespective of diet and genotype. There were also fewer and different biologic responses in males compared to females, as well as different cellular pathways and gene networks (favoring greater neuroprotection in females), together with sex-specific transcription factors and non-protein coding RNAs. Hyperlipidemic stress also resulted in less severe cognitive dysfunction in females. This sex-specific pattern of differential hippocampal microvascular RNA expression might provide therapeutic targets for dementia in males and females.

Long-chain noncoding RNA GAS5 mediates oxidative stress in cardiac microvascular endothelial cells injury.

This study is performed to figure out the role of long-chain noncoding RNA growth-arrest specific transcript 5 (GAS5) in homocysteine (HCY)-induced cardiac microvascular endothelial cells (CMECs) injury. CMECs were cultured and the model of CMECs injury was established by coincubation with HCY. To construct stable overexpression of GAS5 cells, the expression of GAS5, microRNA-33a-5p (miR-33a-5p) and ATP-binding cassette transporter A1 (ABCA1), and biological characteristics of cells were determined. The messenger RNA (mRNA) level and secretion of vascular endothelial growth factor (VEGF), activity of reactive oxygen species (ROS) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) were measured. The binding site between GAS5 and miR-33a-5p and between miR-33a-5p and ABCA1 was verified. CMECs were successfully cultured. Reduction of GAS5 expression and ABCA1 expression together with increased expression of miR-33a-5p was found in CMECs induced by HCY. After overexpression of GAS5, there showed increased proliferative activity, decreased cell apoptosis rate and apoptosis index, enhanced cell migration ability, increased number of lumen formation, increased mRNA expression of VEGF in cells and the secretion in the supernatant, decreased activity of ROS and SOD in cells, and decreased content of ROS in cells. miR-33a-5p could promote the enrichment of GAS5 and ABCA1 was the direct target gene of miR-33a-5p. Our study suggests that the low expression of GAS5 was observed in HCY-induced CMECs injury, and the upregulation of GAS5 could attenuate HCY-induced CMECs injury by mediating oxidative stress, and its mechanism is related to the upregulation of ABCA1 expression by competitively binding with miR-33a-5p.

Tangeretin protects human brain microvascular endothelial cells against oxygen-glucose deprivation-induced injury.

Tangeretin, a citrus flavonoid extracted from the peel of citrus fruits, was reported to possess antiasthmatic, antioxidant, anti-inflammatory, and neuroprotective properties. However, the effect of tangeretin on human brain microvascular endothelial cells (HBMECs) has not been examined. This study was designed to investigate the protective effects of tangeretin on oxygen-glucose deprivation (OGD)-induced injury of HBMECs, and explore the underlying mechanisms. Our results showed that tangeretin improved HBMECs viability in response to OGD. In addition, tangeretin was able to increase the activity of superoxide dismutase and decrease the levels of reactive oxygen species and malondialdehyde (MDA), as well as ameliorate cell apoptosis in OGD-stimulated HBMECs. Mechanistic studies showed that tangeretin prevented the activation of JNK signaling pathway in OGD-stimulated HBMECs. Taken together, our current study demonstrated that tangeretin could ameliorate OGD-induced HBMECs injury through the JNK signaling pathway. Thus, tangeretin might be used as a therapeutic strategy for ischemia-reperfusion brain injury and related diseases.

WNT pathway signaling is associated with microvascular injury and predicts kidney transplant failure.

Microvascular injury is associated with accelerated kidney transplant dysfunction and allograft failure. Molecular pathology can identify new mechanisms of microvascular injury while improving on the diagnostic and prognostic capabilities of traditional histology. We conducted a case-control study of archived kidney biopsy specimens stored up to 10 years with microvascular injury (n = 50) compared with biopsy specimens without histologic injury (n = 45) from patients of similar age, race, and sex. We measured WNT gene expression with a multiplex quantification platform by using digital barcoding, given the importance of WNT reactivation to the response to wounding in the kidney microvasculature and other compartments. Of 210 genes from a commercial WNT panel, 71 were associated with microvascular injury and 79 were associated with allograft failure, with considerable overlap of genes between each set. Molecular pathology identified 46 biopsy specimens with molecular evidence of microvascular injury; 18 (39%) were either C4d negative, donor-specific antibody negative, or had no microvascular injury by histology. The majority of cases with molecular evidence of microvascular injury had poor long-term outcomes. We identified novel WNT pathway genes associated with microvascular injury and allograft failure in residual clinical biopsy specimens obtained up to 10 years earlier. Further mechanistic studies may identify the WNT pathway as a new diagnostic and therapeutic target.

In systemic sclerosis, microvascular and hands digital arteries damage correlates with serum levels of endostatin.

OBJECTIVE: In SSc, vascular injury leads to endothelial dysfunction with reduced capillary blood flow and tissue hypoxia. In SSc, the angiogenesis is impaired and implicated in the microvascular damage. In severe vascular damage, VEGF is reduced and endostatin is increased. The aim of this study was to evaluate the correlation between endostatin serum levels and microvascular and digital arteries damage. METHODS: Seventeen patients with SSc were enrolled in this study. Serum endostatin levels were determined. All patients underwent a NVC, CDUS, and LDPI. RESULTS: The serum level of endostatin significantly (P < .05) increased with NVC progression damage. The mean perfusion significantly (P < .05) decreased with NVC progression damage. Multiple regression analysis showed a significant correlation between endostatin serum level and RI (r = .34, P < .05), PI (r = .60, P < .01), S/D ratio (r = .76, P < .0001), and mean perfusion (r = -.68, P < .001). Endostatin serum levels significantly (P < .05) increased with progression of CDUS damage. CONCLUSIONS: Increased serum endostatin levels are associated with digital vascular damage. In patients with SSc, endostatin is a marker of skin perfusion and digital arteries damage of hands.

Platelets stop us leaking.

In this issue of Blood, Welsh and colleagues determine how platelet thrombi limit the loss of plasma-borne proteins from the microvasculature.

A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature.

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbß3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.

Acute kidney injury is associated with microvascular myocardial damage following myocardial infarction.

Acute kidney injury (AKI) is a frequent complication in patients with ST-elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention. However, the pathophysiology of AKI in this setting is complex and goes beyond the administration of contrast media. Studies assessing the impact of infarct characteristics on AKI are currently lacking. Therefore, we investigated the association of AKI with myocardial as well as microvascular injury in an initial total of 361 consecutive STEMI patients treated by primary percutaneous coronary intervention. Of these, 318 patients were included in final analysis. Serum creatinine was measured on admission as well as 24, 48, and 72 hours thereafter with AKI defined as an increase in serum creatinine of 0.3 mg/dl or more. Cardiac magnetic resonance (CMR) scans were performed in the first week after infarction, with microvascular injury visualized by late gadolinium enhancement CMR defined as any region of hypoenhancement within the hyperenhanced area of infarction. Sixteen patients developed AKI. They showed significantly lower left ventricular ejection fraction (45[interquartile range 40-52]% vs. 54[47-59]%), larger infarct size (21[15-35]% vs. 12[7-22]%) of left ventricular myocardial mass, and more frequent microvascular injury (81 vs. 46%) than those free of AKI. Meaningfully, in multivariate analysis including all CMR data, microvascular injury was the sole independent predictor of AKI (odds ratio 6.74, 95% confidence interval of 1.49-30.43). Thus, among revascularized STEMI patients, the presence of microvascular injury assessed by CMR was independently associated with an increased risk of AKI. This suggests a potential pathophysiological link between cardiac microvascular disease and renal injury following STEMI.

Inflammatory and Angiogenic Factors Linked to Longitudinal Microvascular Changes in Hemodialysis Patients Irrespective of Treatment Dose Intensity.

BACKGROUND: Cardiovascular disease is a major contributor to the poor outcomes observed in hemodialysis. We investigated the relationship between hemodialysis intensity and vascular parameters in high-dose (HDHD; >12hrs/week) and Conventional (CHD; ≤12hrs/week) hemodialysis intensity over a 6-month period. METHODS: We present the 6-month longitudinal analysis of a 2-year multicenter study investigating the effects of HDHD on cardiovascular parameters. We used pulse wave velocity, 24hr ambulatory blood pressure and sublingual dark field capillaroscopy measurements to assess macro- and microcirculation on 6-monthly basis. Pro-inflammatory and endothelial biomarkers were also measured at 6-monthly intervals. RESULTS: 47 participants (21 HDHD, 26 CHD) were studied. CHD were older (63.5±14.2 vs 53.7±12.6 yr; p=0.018), with shorter dialysis vintage (median 23 vs 61 months; p=0.001). There was considerable variability in the degree and direction of change of circulatory measurements over a 6-month period. Hemodialysis intensity (hrs/week) did not correlate to these changes, when adjusted for age, dialysis vintage and comorbidity. Higher levels of Interleukin (IL)-8 measured at baseline independently predicted an increase in the Perfused Boundary Region (5-25µm) of the endothelial glycocalyx (p=0.010) whilst higher levels of soluble Flt-1 had a significant inverse effect (p=0.002) in an adjusted linear model. CONCLUSION: Hemodialysis intensity did not predict changes in either macro- or microvascular parameters. Inflammation mediated through the IL-8 pathway predicted microvascular injury while Flt-1, a potential marker of angiogenesis and endothelial repair, might have a significant protective role. Further understanding of these pathways will be necessary to improve dialysis outcomes.

[The morphological changes in the myocardium associated with the craniocerebral injury]. / Morfologicheskie izmeneniia miokarda pri cherepno-mozgovoi travme.

The objective of the present study was to evaluate the structural changes in the capillaries, arterioles, venules, and cardiomyocytes in the myocardium of the rats following the craniocerebral injury (CCI). Eighteen non-pedigree white female rats with the craniocerebral injury were used as the CCI model. All the animals were given an intraperitoneal injection of sodium thiopental (100 mg/kg b.w.) within 3, 7, and 12 days after the injury. The heart was removed after thoracotomy and the myocardial tissue was examined with the light and electron microscopes. It was shown that the rats with the craniocerebral injury developed well apparent changes in the myocardial tissue during the early post-traumatic period that affected not only the blood vessels themselves (capillaries, arterioles, venules) but also the intra- and extravascular structures. Changes in the microcirculatory system included damages to the mitochondria, myofibrils, cell nuclei, sarcoplasmic reticulum, and cardiomyocytes. It is concluded that the morphological changes in the myocardium of the animals associated with the craniocerebral injury can induce the development of functional disorders in the cardiovascular system during the early post-traumatic period.

(-)-Epigallocatechin Gallate Inhibits Asymmetric Dimethylarginine-Induced Injury in Human Brain Microvascular Endothelial Cells.

(-)-Epigallocatechin gallate (EGCG) is the main polyphenol component of green tea (leaves of the Camellia sinensis plant). EGCG has been reported to protect human brain microvascular endothelial cells (HBMECs) against injury in several models. However, the exact mechanism is still unclear. In the current study we found that EGCG protected against asymmetric dimethylarginine (ADMA)-induced HBMEC injury, and inhibited ADMA-induced reactive oxygen species production and malondialdehyde expression. At the same time, we found that pretreatment with EGCG attenuated the upregulation of Bax and the downregulation of Bcl-2, thus confirming the cellular protective properties of EGCG against ADMA-induced apoptosis. Furthermore, we found that EGCG inhibited ADMA-induced phosphorylation of ERK1/2 and p-38, whose inhibitors relieved HBMEC injury. In conclusion, EGCG can protect against ADMA-induced HBMEC injury via the ERK1/2 and p38 MAPK pathways, which are involved in the underlying mechanisms of HBMEC injury in cerebral infarction.

Zerumbone, a Bioactive Sesquiterpene, Ameliorates Diabetes-Induced Retinal Microvascular Damage through Inhibition of Phospho-p38 Mitogen-Activated Protein Kinase and Nuclear Factor-κB Pathways.

Zerumbone ameliorates retinal damage by blocking advanced glycation end products and their receptor system in streptozotocin-diabetic rats. Because of the multiple factors involved in diabetic retinopathy (DR) etiology, the mechanisms of zerumbone that are mainly responsible for its ameliorative effect on DR need to be further clarified. In the present study, zerumbone (20 mg or 40 mg/kg) or fenofibric acid (100 mg/kg) was orally administered to diabetic rats by intragastric gavage once daily for three consecutive months. Zerumbone displayed similar characteristics to fenofibric acid in reducing retinal vascular permeability and leukostasis in diabetic rats. Fundus photographs showed that large retinal vessel diameters were decreased in zerumbone-treated diabetic rats. Zerumbone not only down-regulated the gene expression of retinal angiogenic parameters, but also reduced the expression of inflammatory cytokines and chemokines in the retina of diabetic rats. Moreover, zerumbone reduced the p38 MAPK phosphorylation and abrogated the nuclear translocation of NF-κB p65 in the retina of diabetic rats. In conclusion, treatment of diabetic rats with zerumbone attenuates the severity of retinal inflammation and angiogenesis, via inhibition of p38 MAPK and NF-κB signaling pathways. These benefits of zerumbone for DR appear to be linked to its antihyperglycemic and antihyperlipidemic effects.

Microvascular injury after lung transplantation.

PURPOSE OF REVIEW: Airway microvessel injury following transplantation has been implicated in the development of chronic rejection. This review focuses on the most recent developments in the field describing preclinical and clinical findings that further implicate the loss of microvascular integrity as an important pathological event in the evolution of irreversible fibrotic remodeling. RECENT FINDINGS: When lungs are transplanted, the airways appear vulnerable from the perspective of perfusion. Two vascular systems are lost, the bronchial artery and the lymphatic circulations, and the remaining vasculature in the airways expresses donor antigens susceptible to alloimmune-mediated injury via innate and adaptive immune mechanisms. Preclinical studies indicate the importance of hypoxia-inducible factor-1α in mediating microvascular repair and that hypoxia-inducible factor-1α can be upregulated to bolster endogenous repair. SUMMARY: Airway microvascular injury is a feature of lung transplantation that limits short-term and long-term organ health. Although some problems are attributable to a missing bronchial artery circulation, another significant issue involves alloimmune-mediated injury to transplant airway microvessels. For a variety of reasons, bronchial artery revascularization surgery at the time of transplantation has not been widely adopted, and the current best hope for this era may be new medical approaches that offer protection against immune-mediated vascular injury or that promote microvascular repair.

Clinical Outcomes Associated With Various Microvascular Injury Patterns Identified by CMR After STEMI.

BACKGROUND: The prognostic significance of various microvascular injury (MVI) patterns after ST-segment elevation myocardial infarction (STEMI) is not well known. OBJECTIVES: This study sought to investigate the prognostic implications of different MVI patterns in STEMI patients. METHODS: The authors analyzed 1,109 STEMI patients included in 3 prospective studies. Cardiac magnetic resonance (CMR) was performed 3 days (Q1-Q3: 2-5 days) after percutaneous coronary intervention (PCI) and included late gadolinium enhancement imaging for microvascular obstruction (MVO) and T2∗ mapping for intramyocardial hemorrhage (IMH). Patients were categorized into those without MVI (MVO-/IMH-), those with MVO but no IMH (MVO+/IMH-), and those with IMH (IMH+). RESULTS: MVI occurred in 633 (57%) patients, of whom 274 (25%) had an MVO+/IMH- pattern and 359 (32%) had an IMH+ pattern. Infarct size was larger and ejection fraction lower in IMH+ than in MVO+/IMH- and MVO-/IMH- (infarct size: 27% vs 19% vs 18% [P < 0.001]; ejection fraction: 45% vs 50% vs 54% [P < 0.001]). During a median follow-up of 12 months (Q1-Q3: 12-35 months), a clinical outcome event occurred more frequently in IMH+ than in MVO+/IMH- and MVO-/IMH- subgroups (19.5% vs 3.6% vs 4.4%; P < 0.001). IMH+ was the sole independent MVI parameter predicting major adverse cardiovascular events (HR: 3.88; 95% CI: 1.93-7.80; P < 0.001). CONCLUSIONS: MVI is associated with future adverse outcomes only in patients with a hemorrhagic phenotype (IMH+). Patients with only MVO (MVO+/IMH-) had a prognosis similar to patients without MVI (MVO-/IMH-). This highlights the independent prognostic importance of IMH in assessing and managing risk after STEMI.

A non-redundant role for MKP5 in limiting ROS production and preventing LPS-induced vascular injury.

There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5(-/-) mice, whereas adoptive transfer of Mkp5(-/-) neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5(-/-) mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47(phox) phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47(phox), and deletion of the p47(phox) gene ablated the LPS-induced vascular injury in Mkp5(-/-) mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury.

Mechanisms of microbubble-vessel interactions and induced stresses: a numerical study.

Oscillating microbubbles within microvessels could induce stresses that lead to bioeffects or vascular damage. Previous work has attributed vascular damage to the vessel expansion or bubble jet. However, ultra-high speed images of recent studies suggest that it could happen due to the vascular invagination. Numerical simulations of confined bubbles could provide insight into understanding the mechanism behind bubble-vessel interactions. In this study, a finite element model of a coupled bubble/fluid/vessel system was developed and validated with experimental data. Also, for a more realistic study viscoelastic properties of microvessels were assessed and incorporated into this comprehensive numerical model. The wall shear stress (WSS) and circumferential stress (CS), metrics of vascular damage, were calculated from these simulations. Resultant amplitudes of oscillation were within 15% of those measured in experiments (four cases). Among the experimental cases, it was numerically found that maximum WSS values were between 1.1-18.3 kPa during bubble expansion and 1.5-74 kPa during bubble collapse. CS was between 0.43-2.2 MPa during expansion and 0.44-6 MPa while invaginated. This finding confirmed that vascular damage could occur during vascular invaginations. Predicted thresholds in which these stresses are higher during vessel invagination were calculated from simulations.