Hydroxysafflor Yellow A Blocks HIF-1α Induction of NOX2 and Protects ZO-1 Protein in Cerebral Microvascular Endothelium

Background: Zonula occludens-1 (ZO-1) protein ensures cerebrovascular integrity against brain ischemic injury. Hydroxysafflor yellow A (HSYA) is a major ingredient of safflower (Carthamus tinctorius L.) with anti-oxidative activity. Because conventional ROS scavengers display poor reactivity with endogenous ROS, this study investigated whether HSYA protected ZO-1 by targeting the enzymes responsible for ROS generation.Methords: Photothrombotic stroke model was prepared in mice to evaluate the protective effect of HSYA on cerebrovascular endothelium. The molecular regulation was investigated in cultured cerebral microvascular endothelial cells (bEnd.3 cells).Results: Oral administration of HSYA (50 mg/kg) reduced cerebral vascular leakage with ZO-1 protection in mice after stroke, largely due to suppression of ROS-associated inflammation. In LPS-stimulated bEnd.3 cells, HSYA increased the ratio of NAD+/NADH to restore Sirt1 induction, which bound to Von Hippel-Lindau (VHL) to ensure HIF-1α protein degradation. Although both NOX1 and NOX2 isoforms were inducible in endothelial cells, we identified NOX2 as the driving force of ROS production. Chromatin immunoprecipitation and luciferase report gene assay revealed that HIF-1α transcriptionally regulated p47phox and Nox2 subunits for the assembly of NOX2 complex, which was blocked by HSYA treatment, largely by reducing HIF-1α accumulation. Inflammation-associated lipid peroxidation impaired ZO-1 protein, but HSYA treatment attenuated carbonyl modification and thus prevented ZO-1 protein from 20S proteasome-mediated degradation, eventually protecting endothelial integrity. In microvascular ZO-1 deficient mice, we further confirmed that HSYA protected cerebrovascular integrity and attenuated ischemic injury dependent on ZO-1 protection. Conclusions: HSYA blocked HIF-1α/NOX2 signaling cascades to protect ZO-1 from proteasomal degradation, suggesting that targeting NOX2 in endothelium is a potential therapeutic strategy to protect against ischemic brain injury.

Clinical case of thrombotic microangiopathy in obstetric practice.

Тромботическая микроангиопатия (ТМА) – клинико-морфологический синдром, в основе которого лежит повреждение эндотелия сосудов микроциркуляторного русла, вызванное разными причинами, но проявляющееся сходной клинической симптоматикой и гистологическими признаками. Одним из важнейших триггеров возникновения ТМА является беременность. Во время беременности возможно развитие вторичной ТМА – при тяжелой преэклампсии и HELLP-синдроме или после тяжелой кровопотери, осложнившейся синдромом диссеминированного внутрисосудистого свертывания крови (ДВС-синдромом). Первый клинический пример иллюстрирует роль в индукции атипичного гемолитико-уремического синдрома (аГУС) многочисленных акушерских осложнений и ДВС-синдрома, возникшего в результате своевременно некомпенсированной кровопотери. Их можно рассматривать как дополнительные комплемент-активирующие состояния. Представленное наблюдение иллюстрирует классическое течение вторичного аГУС с характерными признаками ТМА. Прекращение трансфузий свежезамороженной плазмы (СЗП) и начало таргетной комплемент-блокирующей терапии (экулизумаб) привело к значительному улучшению состояния и обратному развитию ТМА. Во втором наблюдении клинико-лабораторные признаки указывали на наличие вторичной ТМА, вызванной преэклампсией, HELLP-синдромом при отсутствии острого повреждения почек. Назначенная базовая терапия преэклампсии, а также введение СЗП и антикоагулянта позволили прервать внутрисосудистый гемолиз. Thrombotic microangiopathy (TMA) is a clinical morphological syndrome developing as a result of microvascular endothelium damage caused by various reasons but manifesting similar clinical symptoms and histological signs. Pregnancy is one of the most critical TMA triggers. Pregnancy may be accompanied with secondary TMA development in case of severe preeclampsia and HELLP-syndrome or after massive blood loss complicated with disseminated intravascular coagulation (DIC). The first clinical case demonstrates the role of multiple obstetric complications and DIC emerged as a result of failure to timely compensate blood loss in atypical haemolytic-uremic syndrome (aHUS) induction. They may be viewed as additional complement-activating conditions. The described observation illustrates classic progress of secondary aHUS with typical TMA signs. Stopping of fresh frozen plasma (FFP) transfusions and beginning of a target complement blocking therapy (eculizumab) led to significant improvement of condition and TMA involution. In the second observation clinical laboratory signs indicated secondary TMA caused by preeclampsia and HELLP-syndrome without acute renal injury. Prescribed basic therapy of preeclampsia, as well as administration of FFP and anticoagulant, allowed to interrupt intravascular hemolysis.

Wnt Signaling in Inner Blood–Retinal Barrier Maintenance

The retina is a light-sensing ocular tissue that sends information to the brain to enable vision. The blood–retinal barrier (BRB) contributes to maintaining homeostasis in the retinal microenvironment by selectively regulating flux of molecules between systemic circulation and the retina. Maintaining such physiological balance is fundamental to visual function by facilitating the delivery of nutrients and oxygen and for protection from blood-borne toxins. The inner BRB (iBRB), composed mostly of inner retinal vasculature, controls substance exchange mainly via transportation processes between (paracellular) and through (transcellular) the retinal microvascular endothelium. Disruption of iBRB, characterized by retinal edema, is observed in many eye diseases and disturbs the physiological quiescence in the retina’s extracellular space, resulting in vision loss. Consequently, understanding the mechanisms of iBRB formation, maintenance, and breakdown is pivotal to discovering potential targets to restore function to compromised physiological barriers. These unraveled targets can also inform potential drug delivery strategies across the BRB and the blood–brain barrier into retinas and brain tissues, respectively. This review summarizes mechanistic insights into the development and maintenance of iBRB in health and disease, with a specific focus on the Wnt signaling pathway and its regulatory role in both paracellular and transcellular transport across the retinal vascular endothelium.

Resveratrol regulates cytokine secretion by cardiac microvascular endothelium under hypoxia/reoxygenation condition

Background Microvascular endothelial injury is recently considered playing an initial role in myocardial ischemia/reperfusion injury (MIRI). Cardiac microvascular endothelial cells (CMECs) regulate cardiomyocytes and haematocytes via secreting cytokine. MIRI jeopardize not only the barrier function but also the paracrine function of microvasculature. Resveratrol, a natural polyphenolic compound, was demonstrated to protect myocardium against MIRI and to preserve the function of endothelium. However, how the paracrine function of CMECs is regulated by MIRI and resveratrol remains to be elucidated. Purpose The study was to illuminate the alteration of cytokine profiles secreted by CMECs under hypoxia/reoxygenation (H/R) condition and its modulation by resveratrol. Methods CMECs were exposed to different concentrations of resveratrol for 30 minutes and then were subjected to H/R for 12 h/2 h. Apoptotic rates were measured to determine the optimal concentration. Protein antibody arrays were performed to find the alteration of cytokine secreted into conditioned medium by CMECs. A Gene Ontology (GO) analysis was applied to interpret the functional implication of changes in cytokine profiles. Results Resveratrol inhibited apoptosis of CMECs in a dose-dependent manner after H/R and reached its peak effect at the concentration of 100μM, which reduced apoptosis from 27.27±2.95% to 15.01±1.36% (Figure 1A and B). The results of a cluster analysis and all significantly altered factors are shown in figure 1C (fold-change >1.5; p<0.05). Twenty-nine types of cytokine were significantly changed by H/R (15 factors decreased and 14 increased, Figure 2A), and resveratrol at 100μM changed 98 types of cytokine compared with the H/R group (93 factors decreased and 5 increased, Figure 2B). Among these cytokine, eight factors were increased by H/R and they were decreased by resveratrol. Eleven were attenuated by H/R and further decreased by resveratrol. Insulin-like growth factor binding protein-1 was up-regulated by H/R and it was further increased by resveratrol (Figure 2C). The factors with significant alteration were involved in cellular growth, proliferation and differentiation, as well as chemotaxis and transport. Conclusions Resveratrol inhibited the apoptosis of CMECs and modulated the paracrine function of cardiac microvascular endothelium under ischemia/reperfusion condition. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation Figure 1 Figure 2

IMMU-05. A PHASE I STUDY IN PROGRESS OF HEGFRVIII-CD3 BI-SCFV (BRITE) IN PATIENTS WITH WHO GRADE IV MALIGNANT GLIOMA

INTRODUCTION Approximately 30% of glioblastomas harbor the tumor specific EGFRvIII mutation. hEGFRvIII-CD3 bi-scFv (Brain Bi-Specific T Cell Engager - BRiTE) is a novel bispecific antibody construct which can redirect a patient’s entire repertoire of T cells (TCR-agnostic) to specifically lyse EGFRvIII-positive tumor cells. Compared to CAR-T therapy, it offers a highly potent yet off-the-shelf approach for glioblastoma. BRiTE is now entering Phase I clinical trials (NCT04903795) and will be trialed as a monotherapy or with autologous T cell infusion. Migration of T cell binding macromolecules across the blood-brain barrier may be facilitated by activated T cells which adhere to the microvascular endothelium and enter the brain parenchyma. Concurrent administration of activated T cells could therefore enhance trafficking of BRiTE and other antibody-based macromolecules into the intracerebral compartment. HYPOTHESIS We hypothesize that treatment of EGFRvIII-positive WHO grade IV malignant glioma with BRiTE alone or with peripheral T-cell infusion is safe and can induce objective tumor shrinkage at tolerable doses. TRIAL DESIGN/OBJECTIVES A maximum of 18 patients with newly diagnosed or recurrent WHO grade IV malignant EGFRvIII+ glioma will be enrolled after undergoing standard of care treatment. The primary objective will be evaluating the safety of BRiTE alone and with autologous T cell infusion. Patients will receive a single BRiTE infusion, followed 14 days later by an infusion of activated autologous T cells and a second bolus BRiTE infusion. Dose escalation and de-escalation will be managed using a Bayesian optimal interval design. Secondary objectives will be to describe clinical benefit as determined by objective response rate per mRANO criteria, and to evaluate BRiTE pharmacokinetics in serum. CONCLUSION We describe a first-in-human trial of bispecific T cell engager therapy for glioblastoma. We also describe our novel approach for enhancing intracranial penetration of BRiTE using autologous T cell infusion.

SPECT imaging of pulmonary vascular disease in bleomycin-induced lung fibrosis using a vascular endothelium tracer

Background Pulmonary hypertension (PH) complicating idiopathic pulmonary fibrosis (IPF) is associated to worse outcome. There is a great need for a non-invasive diagnostic modality to detect and evaluate the severity of pulmonary vascular disease (PVD). 99mTc-PulmoBind is a novel imaging agent that binds to the adrenomedullin (AM) receptor on the pulmonary microvascular endothelium. SPECT imaging employing the endothelial cell tracer 99mTc-PulmoBind was used to assess PVD associated with lung fibrosis. Methods Rats with selective right lung bleomycin-induced fibrosis were compared to control rats. SPECT imaging was performed after three weeks with 99mTc-PulmoBind and 99mTc-macroaggregates of albumin (MAA). PH and right ventricular (RV) function were assessed by echocardiography. Lung perfusion was evaluated by fluorescent microangiography. Lung AM receptor expression was measured by qPCR and by immunohistology. Relevance to human IPF was explored by measuring AM receptor expression in lung biopsies from IPF patients and healthy controls. Results The bleomycin group developed preferential right lung fibrosis with remodeling and reduced perfusion as assessed with fluorescent microangiography. These rats developed PH with RV hypertrophy and dysfunction. 99mTc-PulmoBind uptake was selectively reduced by 50% in the right lung and associated with reduced AM receptor expression, PH and RV hypertrophy. AM receptor was co-expressed with the endothelial cell protein CD31 in alveolar capillaries, and markedly reduced after bleomycin. Quantitative dynamic analysis of 99mTc-PulmoBind uptake in comparison to 99mTc-MAA revealed that the latter distributed only according to flow, with about 60% increased left lung uptake while left lung uptake of 99mTc-PulmoBind was not affected. Lung from human IPF patients showed important reduction in AM receptor expression closely associated with CD31. Conclusions SPECT imaging with 99mTc-PulmoBind detects PVD and its severity in bleomycin-induced lung fibrosis. Reduced AM receptor expression in human IPF supports further clinical development of this imaging approach.

Mechanisms of Mechanical Force Induced Pulmonary Vascular Endothelial Hyperpermeability

Mechanical ventilation is a supportive therapy for patients with acute respiratory distress syndrome (ARDS). However, it also inevitably produces or aggravates the original lung injury with pathophysiological changes of pulmonary edema caused by increased permeability of alveolar capillaries which composed of microvascular endothelium, alveolar epithelium, and basement membrane. Vascular endothelium forms a semi-selective barrier to regulate body fluid balance. Mechanical ventilation in critically ill patients produces a mechanical force on lung vascular endothelium when the endothelial barrier was destructed. This review aims to provide a comprehensive overview of molecular and signaling mechanisms underlying the endothelial barrier permeability in ventilator-induced lung jury (VILI).

Hydrogen sulfide-dependent microvascular vasodilation is improved following chronic sulfhydryl-donating antihypertensive pharmacotherapy in hypertensive adults.

Hypertension is characterized by systemic microvascular endothelial dysfunction, in part due to a functional absence of hydrogen sulfide (H2S)-mediated endothelium-dependent dilation. Treatment with a sulfhydryl-donating ACE-inhibitor (SH-ACE-inhibitor) improves endothelial function in pre-clinical models of hypertension. To date, no studies have directly assessed the effects of SH-ACE-inhibitor treatment on H2S-dependent vasodilation in hypertensive humans. We hypothesized that SH-ACE-inhibitor treatment would improve H2S-mediated endothelium-dependent vasodilation. Ten hypertensive adults (1W; 56±9yrs, Systolic BP: 141±8.5 mmHg; Diastolic BP: 90.3±6 mmHg) were treated (16 weeks) with the SH-ACE-inhibitor captopril. Red blood cell flux (laser Doppler flowmetry) was measured continuously during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine (ACh; 10-10-10-1 M) alone (control) and in combination with an inhibitor of enzymatic H2S production (10-3 M aminooxyacetate; AOAA) pre- and post-intervention. Cutaneous vascular conductance (CVC; flux/mmHg) was calculated and normalized to the site-specific maximal CVC (0.028M sodium nitroprusside and local heat to 43°C). Area-under-the-curve was calculated using the trapezoid method. The 16-week SH-ACE-inhibitor treatment resulted in a reduction of blood pressure (Systolic BP: 129±10 mmHg; Diastolic BP: 81 ±9 mmHg, both p<0.05). Pre-intervention, inhibition of H2S production had no effect on ACh-induced vasodilation (316±40 control vs 322±35 AU AOAA; p=0.82). Captopril treatment improved ACh-induced vasodilation (316±40 pre vs 399±55 AU post; p=0.04) and increased the H2S-dependent component of ACh-induced vasodilation (pre: -6.6 ± 65.1 vs post: 90.2 ± 148.3 AU, p=0.04). These data suggest that SH-ACE-inhibitor antihypertensive treatment improves cutaneous microvascular endothelium-dependent vasodilation in hypertensive adults, in part via H2S-dependent mechanisms.

Microtubule associated protein 4 (MAP4) phosphorylation reduces cardiac microvascular density through NLRP3-related pyroptosis

Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1β, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.