VEGF Modulates Neurogenesis and Microvascular Remodeling in Epileptogenesis After Status Epilepticus in Immature Rats

Neurogenesis and angiogenesis are widely recognized to occur during epileptogenesis and important in brain development. Because vascular endothelial growth factor (VEGF) is a critical neurovascular target in neurological diseases, its effect on neurogenesis, microvascular remodeling and epileptogenesis in the immature brain after lithium-pilocarpine-induced status epilepticus (SE) was investigated. The dynamic changes in and the correlation between hippocampal neurogenesis and microvascular remodeling after SE and the influence of VEGF or SU5416 injection into the lateral ventricles at different stages after SE on neurogenesis and microvascular remodeling through regulation of VEGF expression were assessed by immunofluorescence and immunohistochemistry. Western blot analysis revealed that the VEGFR2 signaling pathway promotes phosphorylated ERK and phosphorylated AKT expression. The effects of VEGF expression regulation at different stages after SE on pathological changes in hippocampal structure and spontaneous recurrent seizures (SRS) were evaluated by Nissl staining and electroencephalography (EEG). The results showed that hippocampal neurogenesis after SE is related to microvascular regeneration. VEGF promotion in the acute period and inhibition in the latent period after SE alleviates loss of hippocampal neuron, abnormal vascular regeneration and inhibits neural stem cells (NSCs) ectopic migration, which may effectively alleviate SRS severity. Interfering with VEGF via the AKT and ERK pathways in different phases after SE may be a promising strategy for treating and preventing epilepsy in children.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not ‘normoxic’ (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS–MS revealed 52 proteins differentially abundant in sEVs from hypoxic and ‘normoxic’ MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix–receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix–receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Chronic Thromboembolic Pulmonary Hypertension – What Have We Learned From Large Animal Models

Chronic thrombo-embolic pulmonary hypertension (CTEPH) develops in a subset of patients after acute pulmonary embolism. In CTEPH, pulmonary vascular resistance, which is initially elevated due to the obstructions in the larger pulmonary arteries, is further increased by pulmonary microvascular remodeling. The increased afterload of the right ventricle (RV) leads to RV dilation and hypertrophy. This RV remodeling predisposes to arrhythmogenesis and RV failure. Yet, mechanisms involved in pulmonary microvascular remodeling, processes underlying the RV structural and functional adaptability in CTEPH as well as determinants of the susceptibility to arrhythmias such as atrial fibrillation in the context of CTEPH remain incompletely understood. Several large animal models with critical clinical features of human CTEPH and subsequent RV remodeling have relatively recently been developed in swine, sheep, and dogs. In this review we will discuss the current knowledge on the processes underlying development and progression of CTEPH, and on how animal models can help enlarge understanding of these processes.

Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by <i>Saccharomyces boulardii</i>, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Light Sheet Microscopy Using FITC-Albumin Followed by Immunohistochemistry of the Same Rehydrated Brains Reveals Ischemic Brain Injury and Early Microvascular Remodeling

Until recently, the visualization of cerebral microvessels was hampered by the fact that only short segments of vessels could be evaluated in brain sections by histochemistry. These limitations have been overcome by light sheet microscopy, which allows the 3D analysis of microvasculature in cleared brains. A major limitation of light sheet microscopy is that antibodies do not sufficiently penetrate cleared brains. We herein describe a technique of reverse clearing and rehydration, which after microvascular network analysis allows brain sectioning and immunohistochemistry employing a broad set of antibodies. Performing light sheet microscopy on brains of mice exposed to intraluminal middle cerebral artery occlusion (MCAO), we show that in the early phase of microvascular remodeling branching point density was markedly reduced, more strongly than microvascular length. Brain infarcts in light sheet microscopy were sharply demarcated by their autofluorescence signal, closely corresponding to brain infarcts revealed by Nissl staining. Neuronal survival, leukocyte infiltration, and astrocytic reactivity could be evaluated by immunohistochemistry in rehydrated brains, as shown in direct comparisons with non-cleared brains. Immunohistochemistry revealed microthrombi in ischemic microvessels that were likely responsible for the marked branching point loss. The balance between microvascular thrombosis and remodeling warrants further studies at later time-points after stroke.

Abstract 16948: A Comprehensive and High Accurate Pipeline for Mouse Retinal Vasculature Characterization

Rationale: Vascular morphological features reflect the vessel biological functions and analysis of these features is critical for understanding physiological and pathological process of vascular development and vascular diseases. Mouse retinal vasculature is a well-recognized and commonly used animal model for angiogenesis and microvascular remodeling. Thus, a powerful tool to analyze morphological changes of retinal vasculature is in urgent need. Objective: Develop a comprehensive and high accurate pipeline to analyze morphological changes of mouse retinal vasculature. Methods and Results: Retinas from postnatal mice at P2-P7 are harvested and retinal vasculatures are visualized by isolectin B4 whole mount staining. A comprehensive software, Vessel Tech, is developed to perform vessel segmentation, network reconstruction and analysis. Vessel Tech relies on recent advancements in the convolutional neural network and techniques from network data analysis to provide high accuracy vascular identification and comprehensive vascular morphological feature extraction. Compared with existing blood vessel analysis softwares, Vessel Tech shows great improvement in accuracy. Based on the reconstructed vessel network, various metrics of angiogenesis and micro-remodeling are extracted and statistically analyzed. Conclusions: We generated and verified a novel pipeline, named “Vessel Tech”, which could automatically process retinal vascular images, reconstruct vessel network with high accuracy and assay global and local vascular characteristics. The development of Vessel Tech provides a powerful tool to precisely study the physiological and pathological variations during vascular development and vascular diseases.

Coronary flow evaluation in heart transplant patients compared to healthy controls documents the inadequacy of the coronary flow velocity reserve metric

Background Coronary microvasculopathy has impact on prognosis in heart transplantation (HT). Distinct contributions by functional or structural alterations of coronary microcirculation in HT and their prognostic role have not been fully elucidated. Purpose We aimed to identify the mechanisms of coronary microvascular impairment in HT and their possible prognostic implications by applying a comprehensive analysis in a comparative study. Methods Included were 134 patients, surviving at least 5 years, with normal systolic function and no evidence of allograft vasculopathy or symptoms/signs of rejection. To permit comparison, 50 healthy volunteers without cardiovascular diseases, and matched for age and sex, served as controls. All underwent echocardiographic evaluation of microvascular function by the assessment of rest and hyperemic diastolic peak blood velocity (DPVr and DPVh). These paired data enable calculation of coronary flow velocity reserve (CFVR) and its inherent companion that is based on the quadratic mean: CFVRC = √{(DPVr)2 + (DPVh)2}. Additionally, basal and hyperemic coronary microvascular resistance (BMR and HMR) were estimated. A CFVR ≤2.5 was considered abnormal; the median value of DPVh (75 cm/s) and CFVRC (80 cm/s) were selected as cut-offs to classify patients. Results HT patients can be assigned to four groups, based on their CFVR and DPVh (Figure A): group 1 (n=32), discordant with preserved CFVR (3.1±0.4); group 2 (n=60), concordant with preserved CFVR (3.4±0.5); group 3 (n=31), concordant with impaired CFVR (1.8±0.3) and group 4 (n=11), discordant with impaired CFVR (2.0±0.2). Group 3 represents the structural microvascular remodeling with high HMR, while group 4 represents the functional remodeling with low BMR. Intriguingly, group 1 showed lower DPVr (p&lt;0.0001) and lower DPVh (p&lt;0.0001) than controls (Figure B, upper panel) with lower CFVR (p&lt;0.0001), even if normal, and lower CFVRC (p&lt;0.0001) than controls (Figure B, lower panel). Moreover, both BMR and HMR were higher in group 1 than in controls (5.3±1 vs 4.4±1.2, p=0.001 and 1.5±0.3 vs 1.1±0.2, p&lt;0.0001, respectively), suggesting structural microvascular remodeling. Conversely, group 2 was comparable with controls (Figure B). Clinical characteristics of the different groups are shown in the Table. 13/32 (40.6%) patients in group 1 died in a follow up of 28 years and mortality rate was comparable to group 3 (14/31, 45.2%). However, CFVRC was &lt;80 cm/s in all 13 deaths in group 1, yet being characterized by preserved CFVR (Figure C). Conclusions A normal CFVR could hide detection of microvascular damage with high flow resistance and low flow velocities at rest. This microvasculopathy seems to be secondary to factors unrelated to HT (i.e., less rejections and more often diabetes). Being a dimensionless ratio, CFVR may miss some deaths, yet captured by CFVRC. Thus, the combined use of CFVR and CFVRC provides more complete clinical information on coronary microvasculopathy in HT. Funding Acknowledgement Type of funding source: None

Hypoxia induced interstitial transformation of microvascular endothelial cells by mediating HIF-1α/VEGF signaling in systemic scleroderma

Background: Endothelial mesenchymal transition (EndMT) is a key pathological event for vasculopathy, and is one of the early features and hallmarks of systemic scleroderma (SSc). It has been well-established that hypoxia contributes to EndMT. However, little is known about the effects of EndMT induced by hypoxia on the skin microvascular remodeling of SSc, as well as the underlying mechanism. Methods: Skin biopsy was performed for SSc patients and healthy controls, and skin tissues were collected for isobaric tags for the relative and absolute quantification (iTRAQ)-based proteomics and immunohistochemical test. Human microvascular endothelial cell line-1 (HMEC-1) cultured in hypoxic or normal conditions was treated by tamoxifen or bevacizumab. The expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF)-a, CD31, α-smooth muscle actin (α-SMA), VE-cadherin, and fibronectin were detected at both the protein and mRNA level.Results: The iTRAQ-based proteomics indicated the significantly upregulated HIF-1 signal in the skin tissues of SSc patients. The immunohistochemical results demonstrated the significant downregulation of endothelial cell (EC) marker CD31 and the distinct positive staining of interstitial cell (IC) marker α-SMA at sites that lined the vessel lumens in skin tissues of SSc. Meanwhile, the positive staining of HIF-1α, which is a key transcription factor in response to chronic hypoxia, and VEGF-a were found to be diffusely distributed in SSc skin tissues. Consistent with these observations, HMEC-1 cells cultured under hypoxic conditions exhibited a significant decrease in CD31 and VE-cadherin expression, alongside the marked increase in the expression of α-SMA and fibronectin, as well as the distinct upregulation of HIF-1α and VEGF-a, when compared with those under normal conditions. It is noteworthy that the inhibition of HIF-1α by tamoxifen effectively downregulated the hypoxic induction of VEGF-a and α-SMA, while rescuing the hypoxic suppression of CD31. In addition, the VEGF-a inhibitor bevacizumab treatment had the same effect on the hypoxic expression of α-SMA and CD31, as a tamoxifen intervention, but did not reduce HIF-1α. Conclusion: These results suggest that the HIF-1α/VEGF signaling pathway has a critical role in mediating the effect of hypoxia-induced EndMT on the skin microvascular remodeling of SSc.