Pericyte, but not astrocyte, hypoxia inducible factor-1 (HIF-1) drives hypoxia-induced vascular permeability in vivo

Background Ways to prevent disease-induced vascular modifications that accelerate brain damage remain largely elusive. Improved understanding of perivascular cell signalling could provide unparalleled insight as these cells impact vascular stability and functionality of the neurovascular unit as a whole. Identifying key drivers of astrocyte and pericyte responses that modify cell–cell interactions and crosstalk during injury is key. At the cellular level, injury-induced outcomes are closely entwined with activation of the hypoxia-inducible factor-1 (HIF-1) pathway. Studies clearly suggest that endothelial HIF-1 signalling increases blood–brain barrier permeability but the influence of perivascular HIF-1 induction on outcome is unknown. Using novel mouse lines with astrocyte and pericyte targeted HIF-1 loss of function, we herein show that vascular stability in vivo is differentially impacted by perivascular hypoxia-induced HIF-1 stabilization. Methods To facilitate HIF-1 deletion in adult mice without developmental complications, novel Cre-inducible astrocyte-targeted (GFAP-CreERT2; HIF-1αfl/fl and GLAST-CreERT2; HIF-1αfl/fl) and pericyte-targeted (SMMHC-CreERT2; HIF-1αfl/fl) transgenic animals were generated. Mice in their home cages were exposed to either normoxia (21% O2) or hypoxia (8% O2) for 96 h in an oxygen-controlled humidified glove box. All lines were similarly responsive to hypoxic challenge and post-Cre activation showed significantly reduced HIF-1 target gene levels in the individual cells as predicted. Results Unexpectedly, hypoxia-induced vascular remodelling was unaffected by HIF-1 loss of function in the two astrocyte lines but effectively blocked in the pericyte line. In correlation, hypoxia-induced barrier permeability and water accumulation were abrogated only in pericyte targeted HIF-1 loss of function mice. In contrast to expectation, brain and serum levels of hypoxia-induced VEGF, TGF-β and MMPs (genes known to mediate vascular remodelling) were unaffected by HIF-1 deletion in all lines. However, in agreement with the permeability data, immunofluorescence and electron microscopy showed clear prevention of hypoxia-induced tight junction disruption in the pericyte loss of function line. Conclusion This study shows that pericyte but not astrocyte HIF-1 stabilization modulates endothelial tight junction functionality and thereby plays a pivotal role in hypoxia-induced vascular dysfunction. Whether the cells respond similarly or differentially to other injury stimuli will be of significant relevance.

Abstract P408: Mesenchymal Stem Cell-derived Angptl4 Improves Endothelial Permeability And Resolution Of Inflammation In Atherosclerosis

Objective: Given the fundamental contribution of inflammation to atherosclerosis, we studied the effect of ANGPTL4 in regulating vascular lesions during atherosclerosis. Methods and Results: We analyzed plasma levels of ANGPTL4 and found that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than did patients with lower ANGPTL4 levels ( p <0.05). Moreover, in AMI patients with heart failure (HF) at admission, the recurrence of HF was lower in patients with a higher level of ANGPTL4. We then investigated the therapeutic application of ANGPTL4 in an atherosclerosis model. Apoe-/- mice fed a high-fat diet were injected with PBS or ANGPTL4 protein (2 μg per mouse, i.p. ) three times per week for 7 weeks. En face staining and mRNA analysis showed that plaque size, necrotic core area, lipid accumulation, and inflammatory molecules were greatly reduced in the ANGPTL4 group. Aortic permeability, measured by leakage of Evans blue dye, was significantly decreased in the ANGPTL4 group. The induction of pro-inflammatory mediators was significantly inhibited in endothelial cells, vascular smooth muscle cells, and macrophages by ANGPTL4 treatment. Endothelial Krüppel-like factor 2 (KLF2) and VE-cadherin were restored to contribute to maintenance of vascular integrity by ANGPTL4 treatment. Elevated levels of circulating leptin, interleukin-6, and interleukin-1β were profoundly reduced. Most importantly, the fibrous cap was significant thicker in the ANGPTL4 group than in the PBS group. Conclusions: ANGPTL4 treatment attenuated atherogenesis, which suggests that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. More importantly, ANGPTL4 treatment inhibits necrotic core formation in lesions, leading to the formation of more stable plaques as evidenced by increased fibrous cap thickness.

Angiopoietins as Targets for Diabetic Retinopathy Treatment

Diabetic eye diseases, such as diabetic retinopathy (DR) and diabetic macular edema (DME) are among the leading causes of blindness in developed countries. Anti-VEGF therapies such as, ranibizumab, aflibercept and off-label bevacizumab have become first-line treatment for DME. While randomized controlled trials show significant improvement in vision, these anti-VEGF agents have limited durability leading to a significant treatment burden, as reflected in real-world studies, which generally demonstrate under-treatment and less favorable visual acuity outcomes than observed in prospective trials. Alternative pathways, such as the Tie-2 angiopoietin pathway may address unmet needs, with potential for greater efficacy or durability when compared to anti-VEGF monotherapy. While some Tie-2 angiopoietin therapeutic agents, such as nesvacumab, ARP-1536 or AKB-9778, did not meet primary endpoints in clinical trials, other agents have shown promise. One such agent is faricimab, a bispecific antibody inhibiting both VEGF-A and Ang-2. The phase 3 DME trials (YOSEMITE and RHINE) demonstrated favorable safety, visual, and durability outcomes; patients receiving faricimab injection every 4 months achieved similar visual gains as those receiving aflibercept injection every 2 months. Another agent, AXT107 is a peptide that inhibits VEGFR2 and modifies Ang-2 to behave more similarly to Ang-1, promoting vascular stability. This drug is currently undergoing phase 1/2a trials for safety and bioactivity to be completed in May 2022.

Cell Metabolomics Responses to Chlorpromazine: Novel Insights Into Blood Brain Barrier Dysfunctionality

Blood-brain barrier (BBB) prevents drug access and impedes therapeutic efficacy. Effective methods of modulating barrier function and resolving these difficulties are desperately needed. We hypothesized that cell metabolic adaptation significantly influences physiological and pathological barrier functionality because we were convinced that a better understanding of cell-oriented BBB responses could provide valuable insight, and because metabolic dysregulation is prominent in many vascular-related pathological processes associated with BBB disruption. Biochemical fingerprints of primary brain endothelial cells (EC) were obtained using untargeted liquid chromatography–mass spectrometry (LC-MS) metabolomic profiling during chlorpromazine treatment, at concentration similar to its measured in the serum of high dosed patients, providing a functional readout of cell status. Metabolomic analyses showed brain EC had a distinct metabolic signature. Corroborating their role in BBB and CNS protection. Surprisingly, EC largely maintained their normoxic characteristics in drug treatment situations and their profiles diverged from those of control. When examining EC reactions, tissue specificity/origin is definitely significant. With a focus on cellular metabolism, we examine how cell metabolic adaptive capabilities may influence vascular stability, as well as the prospect that modifying metabolite levels may be an efficient strategy to modulate brain EC function. Overall, this study sheds new light on cell-associated metabolic changes and serves as a valuable resource for understanding BBB modifications in drug treatment circumstances.

RNF213 and GUCY1A3 in Moyamoya Disease: Key Regulators of Metabolism, Inflammation, and Vascular Stability

Moyamoya disease is an idiopathic chronically progressive cerebrovascular disease, which causes both ischemic and hemorrhagic stroke. Genetic studies identified RNF213/Mysterin and GUCY1A3 as disease-causing genes. They were also known to be associated with non-moyamoya intracranial large artery disease, coronary artery disease and pulmonary artery hypertension. This review focused on these two molecules and their strong linker, calcineurin/NFAT signaling and caveolin to understand the pathophysiology of moyamoya disease and related vascular diseases. They are important regulators of lipid metabolism especially lipotoxicity, NF-κB mediated inflammation, and nitric oxide-mediated vascular protection. Although intimal thickening with fibrosis and damaged vascular smooth muscle cells are the distinguishing features of moyamoya disease, origin of the fibrous tissue and the mechanism of smooth muscle cell damages remains not fully elucidated. Endothelial cells and smooth muscle cells have long been a focus of interest, but other vascular components such as immune cells and extracellular matrix also need to be investigated in future studies. Molecular research on moyamoya disease would give us a clue to understand the mechanism preserving vascular stability.

Angiopoietin/Tie2 signalling and its role in retinal and choroidal vascular diseases: a review of preclinical data

The angopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Ang/Tie) pathway is an emerging key regulator in vascular development and maintenance. Its relevance to clinicians and basic scientists as a potential therapeutic target in retinal and choroidal vascular diseases is highlighted by recent preclinical and clinical evidence. The Ang/Tie pathway plays an important role in the regulation of vascular stability, in angiogenesis under physiological and pathological conditions, as well as in inflammation. Under physiological conditions, angiopoietin-1 (Ang-1) binds to and phosphorylates the Tie2 receptor, leading to downstream signalling that promotes cell survival and vascular stability. Angiopoietin-2 (Ang-2) is upregulated under pathological conditions and acts as a context-dependent agonist/antagonist of the Ang-1/Tie2 axis, causing vascular destabilisation and sensitising blood vessels to the effects of vascular endothelial growth factor-A (VEGF-A). Ang-2 and VEGF-A synergistically drive vascular leakage, neovascularisation and inflammation, key components of retinal vascular diseases. Preclinical evidence suggests that modulating the Ang/Tie pathway restores vascular stabilisation and reduces inflammation. This review discusses how targeting the Ang/Tie pathway or applying Ang-2/VEGF-A combination therapy may be a valuable therapeutic strategy for restoring vascular stability and reducing inflammation in the treatment of retinal and choroidal vascular diseases.

3D curvature-instructed endothelial flow response and tissue vascularization

Vascularization remains a long-standing challenge in engineering complex tissues. Particularly needed is recapitulating 3D vascular features, including continuous geometries with defined diameter, curvature, and torsion. Here, we developed a spiral microvessel model that allows precise control of curvature and torsion and supports homogeneous tissue perfusion at the centimeter scale. Using this system, we showed proof-of-principle modeling of tumor progression and engineered cardiac tissue vascularization. We demonstrated that 3D curvature induced rotation and mixing under laminar flow, leading to unique phenotypic and transcriptional changes in endothelial cells (ECs). Bulk and single-cell RNA-seq identified specific EC gene clusters in spiral microvessels. These mark a proinflammatory phenotype associated with vascular development and remodeling, and a unique cell cluster expressing genes regulating vascular stability and development. Our results shed light on the role of heterogeneous vascular structures in differential development and pathogenesis and provide previously unavailable tools to potentially improve tissue vascularization and regeneration.

New Soluble Angiopoietin Analog of C4BP-ANG1 Prevents Pathological Vascular Leakage

Vascular leak is a key driver of organ injury in diseases such as Acute Respiratory Distress Syndrome caused by viruses, including COVID-19. Strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the Angiopoietin-1 (Angpt1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANGPT1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANGPT1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α (C4BP). We refer to this new fusion protein biologic as C4BP-ANG1, which forms a stable heptamer and induces TIE2 phosphorylation in cultured cells, and in the lung following i.v. injection of mice. Injection of C4BP-ANG1 ameliorates VEGF- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability, and therefore is a promising candidate treatment for inflammatory endothelial dysfunction.