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.

Evaluation of the Biocompatibility and Endothelial Differentiation Capacity of Mesenchymal Stem Cells by Polyethylene Glycol Nanogold Composites

Cardiovascular Diseases (CVDs) such as atherosclerosis, where inflammation occurs in the blood vessel wall, are one of the major causes of death worldwide. Mesenchymal Stem Cells (MSCs)-based treatment coupled with nanoparticles is considered to be a potential and promising therapeutic strategy for vascular regeneration. Thus, angiogenesis enhanced by nanoparticles is of critical concern. In this study, Polyethylene Glycol (PEG) incorporated with 43.5 ppm of gold (Au) nanoparticles was prepared for the evaluation of biological effects through in vitro and in vivo assessments. The physicochemical properties of PEG and PEG–Au nanocomposites were first characterized by UV-Vis spectrophotometry (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), and Atomic Force Microscopy (AFMs). Furthermore, the reactive oxygen species scavenger ability as well as the hydrophilic property of the nanocomposites were also investigated. Afterwards, the biocompatibility and biological functions of the PEG–Au nanocomposites were evaluated through in vitro assays. The thin coating of PEG containing 43.5 ppm of Au nanoparticles induced the least platelet and monocyte activation. Additionally, the cell behavior of MSCs on PEG–Au 43.5 ppm coating demonstrated better cell proliferation, low ROS generation, and enhancement of cell migration, as well as protein expression of the endothelialization marker CD31, which is associated with angiogenesis capacity. Furthermore, anti-inflammatory and endothelial differentiation ability were both evaluated through in vivo assessments. The evidence demonstrated that PEG–Au 43.5 ppm implantation inhibited capsule formation and facilitated the expression of CD31 in rat models. TUNEL assay also indicated that PEG–Au nanocomposites would not induce significant cell apoptosis. The above results elucidate that the surface modification of PEG–Au nanomaterials may enable them to serve as efficient tools for vascular regeneration grafts.

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Astragaloside IV Inhibits Bleomycin-Induced Ferroptosis in Human Umbilical Vein Endothelial Cells by Mediating LPC

Ferroptosis, as an iron-dependent programmed cell death pathway, can induce a variety of cardiovascular diseases. Astragaloside IV (AS-IV), which is purified from Astragalus membranaceus, can protect endothelial function and promote vascular regeneration. However, the role played by AS-IV in ferroptosis remains unknown. In this study, the lipid metabolomics in HUVECs treated with/without bleomycin and/or AS-IV were explored using LC/MS. The most differential metabolite between groups was further identified via GO and pathway enrichment analyses. The effects of lysophosphatidylcholine (LPC), AS-IV, and FIN56 on cell viability were explored using the CCK-8 assay, their effects on cell senescence were examined by β-galactosidase staining, and their effects on ferroptosis were detected by a flow cytometric analysis of lipid ROS levels, transmission electron microscopy, and an assay for cellular iron levels. The related mechanisms were investigated by real-time PCR and Western blot assays. Our results showed that LPC, as the most differential metabolite, inhibited cell viability but promoted cell apoptosis and senescence as its concentration increased. Also, the decreased cell activity, increased iron ion and lipid ROS levels, and the enhanced cell senescence induced by LPC treatment were all significantly reversed by AS-IV but further enhanced by FIN56 treatment. The changes in mitochondrial morphology caused by the LPC treatment were significantly alleviated by the AS-IV treatment, while treatment with FIN56 reversed those phenomena. Moreover, AS-IV partially upregulated the levels of SLC7A11 and GPX4 expression which were reduced by LPC. However, those changes were prevented by FIN56 treatment. In conclusion, our data suggested that AS-IV could serve as a novel drug for treating ferroptosis-related diseases.

The SARS-CoV-2 Spike protein alters human cardiac pericyte function and interaction with endothelial cells through a non-infective mechanism involving activation of CD147 receptor signalling

Background Human cardiac pericytes (PC) were proposed as the main cellular target for SARS-CoV-2 in the heart due to high transcriptional levels of the angiotensin-converting enzyme 2 (ACE2) receptor. Emerging reports indicate CD147/Basigin (BSG), highly expressed in endothelial cells (EC), is an alternative SARS-CoV-2 receptor. To date, the mechanism by which the virus infects and disrupts the heart vascular cells was not identified yet. Moreover, cleaved Spike (S) protein molecules could be released into the bloodstream from the leaking pulmonary epithelial-endothelial barrier in patients with severe COVID-19, opening to the possibility of non-infective diseases in organs distant from the primary site of infection. Purposes (1) to confirm that human primary cardiac PC express ACE2 and CD147; (2) to verify if PC are permissible to SARS-CoV-2 infection; (3) to investigate if the recombinant SARS-CoV-2 S protein alone, without the other viral elements, can trigger molecular signalling and induce functional alterations in PC; (4) to explore which viral receptor is responsible for the observed events. Methods and results Cardiac PC express both the ACE2 and CD147 receptors at mRNA and protein level. Incubation of PC for up to 5 days with SARS-CoV-2 expressing the green fluorescent protein (GFP) did not show any evidence of cell infection or viral replication. Next, we exposed the PC to the recombinant S protein (5.8 nM) and confirmed that the protein engaged with cellular receptors (western blot analysis of S protein in treated and control PC). Incubation with the S protein increased PC migration (wound closure assay, P<0.01 vs ctrl) and reduced the formation of tubular structures between PC and EC in a Matrigel assay (P<0.01 vs ctrl). Moreover, the S protein promoted the production of pro-inflammatory factors typical of the cytokine storm in PC (ELISA measurement of MCP1, IL-6, IL-1β, TNFα, P<0.05 vs ctrl), and induced the secretion of pro-apoptotic factors responsible for EC death (Caspase 3/7 assay, P<0.05 vs ctrl). Signalling studies revealed that the S protein triggers the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in cardiac PC. The neutralization of CD147, using a blocking antibody, prevented ERK1/2 activation in PC, and was reflected into a partial rescue of the cell functional behaviour (migration and pro-angiogenic capacity). In contrast, blockage of CD147 failed to prevent the pro-inflammatory response in PC. Conclusions We propose the novel hypothesis that COVID-19 associated heart's microvascular dysfunction is prompted by circulating S protein molecules rather than by the direct coronavirus infection of PC. Besides, we propose CD147, and not ACE2, as the leading receptor mediating S protein signalling in cardiac PC. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): BHF project grant “Targeting the SARS-CoV-2 S-protein binding to the ACE2 receptor to preserve human cardiac pericytes function in COVID-19” BHF Centre for Vascular Regeneration II

Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury

Background Coexistent chronic kidney diseases (CKD) and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway. Methods To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients. Results We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 of monocytes in vitro. In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events. Conclusions Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.

Pre-clinical Research of Human Amnion-derived Mesenchymal Stem Cells and its First Clinical Treatment for a Severe Uremic Calciphylaxis Patient

Calciphylaxis is a rare disease characterized histologically by microvessel calcification and microthrombosis, with high mortality and no proven therapy. We reported a severe uremic calciphylaxis patient with progressive skin ischemia, large areas of painful malodorous ulcers and mummified legs. Because of her rapid progression and refractory to conventional therapy, human amnion-derived mesenchymal stem cells (hAMSCs) treatment was approved. Establishment and release inspection of hAMSCs, efficacy and safety assessment including cytokines secretory ability, immunocompetence, tumorigenicity and genetics analysis in vitro were introduced. We further performed acute and long-term hAMSC toxity evaluations in C57BL/6 mice/rats, abnormal immune response tests in C57BL/6 mice and tumorigenic tests in the neonatal NU nude mice. After pre-clinical research, she was treated by hAMSCs with intravenous and local intramuscular injection and external supernatants application to her ulcers. When followed up to 15 months, her blood-based markers of bone and mineral metabolism were improved, with regeneration of skin soft tissue and a more favorable profile of peripheral blood mononuclear cells. Skin biopsy after 1 month treatment showed vascular regeneration with mature non-calcified vessels within dermis and 20 months later re-epithelialization restored the integrity of damaged site. No infusion or local treatment related adverse events occurred. To the best of our knowledge, this is the first evidence for the clinical use of hAMSCs. These findings suggest hAMSCs warrant further investigation as a potential regenerative treatment for uremic calciphylaxis with effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, anti-inflammatory and immune modulation, multi-differentiation, re-epithelialization and restorage of integrity.