Cerebral Angiogenesis Research Articles

Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood-brain barrier integrity

The neurovascular unit (NVU) is a complex multicellular structure that helps maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration, the underlying molecular mechanisms remain unclear. Here, we use zebrafish embryos carrying a mutation in Scavenger Receptor B2, a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells (RGCs), to investigate the interplay among different NVU components. Through live imaging and genetic manipulations, we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling, thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling, leading to severe vascular defects, hemorrhages, and a leaky BBB. Altogether, our findings provide insights into NVU formation and function and offer avenues for investigating diseases involving white matter defects and vascular abnormalities.

Acid sphingomyelinase inhibition induces cerebral angiogenesis post-ischemia/reperfusion in an oxidative stress-dependent way and promotes endothelial survival by regulating mitochondrial metabolism

Acid sphingomyelinase (ASM) inhibitors are widely used for the treatment of post-stroke depression. They promote neurological recovery in animal stroke models via neurorestorative effects. In a previous study, we found that antidepressants including amitriptyline, fluoxetine, and desipramine increase cerebral angiogenesis post-ischemia/reperfusion (I/R) in an ASM-dependent way. To elucidate the underlying mechanisms, we investigated the effects of the functional ASM inhibitor amitriptyline in two models of I/R injury, that is, in human cerebral microvascular endothelial hCMEC/D3 cells exposed to oxygen-glucose deprivation and in mice exposed to middle cerebral artery occlusion (MCAO). In addition to our earlier studies, we now show that amitriptyline increased mitochondrial reactive oxygen species (ROS) formation in hCMEC/D3 cells and increased ROS formation in the vascular compartment of MCAO mice. ROS formation was instrumental for amitriptyline’s angiogenic effects. ROS formation did not result in excessive endothelial injury. Instead, amitriptyline induced a profound metabolic reprogramming of endothelial cells that comprised reduced endothelial proliferation, reduced mitochondrial energy metabolism, reduced endoplasmic reticulum stress, increased autophagy/mitophagy, stimulation of antioxidant responses and inhibition of apoptotic cell death. Specifically, the antioxidant heme oxygenase-1, which was upregulated by amitriptyline, mediated amitriptyline’s angiogenic effects. Thus, heme oxygenase-1 knockdown severely compromised angiogenesis and abolished amitriptyline’s angiogenic responses. Our data demonstrate that ASM inhibition reregulates a complex network of metabolic and mitochondrial responses post-I/R that contribute to cerebral angiogenesis without compromising endothelial survival.

Carotid artery transplantation of brain endothelial cells enhances neuroprotection and neurorepair in ischaemic stroke rats.

Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.

RESWT promoted angiogenesis via Bach1/Wnt/β-catenin signaling pathway.

Previous reports have established that rESWT fosters angiogenesis, yet the mechanism by which rESWT promotes cerebral angiogenesis remains elusive. rESWT stimulated HUVECs proliferation as evidenced by the CCK-8 test, with an optimal dosage of 2.0 Bar, 200 impulses, and 2Hz. The tube formation assay of HUVECs revealed that tube formation peaked at 36h post-rESWT treatment, concurrent with the lowest expression level of Bach1, as detected by both Western blot and immunofluorescence. The expression level of Wnt3a, β-catenin, and VEGF also peaked at 36h. A Bach1 overexpression plasmid was transfected into HUVECs, resulting in a decreased expression level of Wnt3a, β-catenin, and VEGF. Upon treatment with rESWT, the down-regulation of Wnt3a, β-catenin, and VEGF expression in the transfected cells was reversed. The Wnt/β-catenin inhibitor DKK-1 was utilized to suppress Wnt3a and β-catenin expression, which led to a concurrent decrease in VEGF expression. However, rESWT treatment could restore the expression of these three proteins, even in the presence of DKK-1. Moreover, in the established OGD model, it was observed that rESWT could inhibit the overexpression of Bach1 and enhance VEGF and VEGFR-2 expression under the OGD environment.

Role of L-PGDS in Light Alcohol Consumption-Induced Cerebral Angiogenesis in Male Mice

Ischemic stroke is one of the leading causes of death and permanent disability. Promoting cerebral angiogenesis before or after ischemic stroke reduces ischemic brain damage and improves functional recovery. Alcohol is one of the most commonly consumed chemical substances. Light alcohol consumption (LAC) reduces the incidence and improves the prognosis of ischemic stroke. Recently, we found that LAC promotes cerebral angiogenesis under basal conditions and following ischemic stroke. In addition, LAC upregulates lipocalin-type prostaglandin D2 synthase (L-PGDS), the principal PGH2 isomerase mainly expressed in the central nervous and cardiovascular systems. Therefore, we determined the role of L-PGDS in LAC-induced cerebral angiogenesis. In in vitro studies, low-concentration ethanol significantly upregulated L-PGDS and increased angiogenic capability (tube formation, migration, and proliferation) in cultured C57BL/6J mouse brain microvascular endothelial cells (MBMVECs). AT-56, a selective L-PGDS inhibitor, abolished the increased angiogenic capability. In in vivo studies, 8-week gavage feeding with low-dose ethanol significantly upregulated L-PGDS and increased vessel density and vessel branches in the cerebral cortex and subcortical area under physiological conditions and following ischemic stroke in C57BL/6J male mice. AT-56 and endothelial cell-specific L-PGDS conditional knockout did not affect vessel density and vessel branches of the cerebral cortex and subcortical area in water-fed control mice but alleviated the proangiogenic effect in low-dose ethanol-fed mice under basal conditions and following ischemic stroke. Therefore, our findings suggest that L-PGDS may be crucial in LAC-induced cerebral angiogenesis. NIH 2R01AA023610. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Preconditioning exercise reduces brain damage of ischemic stroke in rats via PI3K-AKT pathway by bioinformatic analysis.

Ischemic stroke is one of the most vital causes of high neurological morbidity and mortality in the world. Preconditioning exercise is considered as the primary prevention of stroke to resistance to subsequent injury. We tried to research the underlying biological mechanisms of this exercise. Forty-two SD rats were randomly divided into three groups: middle cerebral artery occlusion (MCAO) group, exercise group with MCAO (EX + MCAO) group, and sham group, with 14 rats in each group. The EX + MCAO group underwent exercise preconditioning for 3weeks before occlusion, and the other two groups were fed and exercised normally. After 3weeks, MCAO model was made by thread plug method in the EX + MCAO group and MCAO group. After successful modeling, the Longa scale was used to evaluate the neurological impairment of rats at day 0, day 1, and day 2. The rats in each group were killed on the third day after modeling. TTC staining measured the infarct volume of each group. The morphology and apoptosis of cortical cells were observed by HE and Tunel staining. Three rats in each group underwent high-throughput sequencing. Bioinformatic analysis was used to find the deferentially expressed genes (DEGs) and predict the transcription factor binding sites (TFBS) of the next-generation sequencing results. Gene enrichment (GSEA) was used to analyze potential functional genes and their corresponding signaling pathways. The Longa scale showed EX + MCAO group had the neurological function better than the modeling group (P < 0.001). TTC staining showed that the infarct size of EX + MCAO group was less than MCAO group (P < 0.05). HE and Tunel staining showed that the cells in the EX + MCAO group and the sham group had normal morphology and fewer apoptotic cells than MCAO group. A new gene named 7994 was discovered and TFBS of this gene was predicted, which could interact with key genes such as Foxd3, Foxa2, NR4A2, SP1, CEBPA, and SOX10. GSEA showed that EX + MCAO group could promote and regulate angiogenesis and apoptosis through PI3K-AKT pathway. Preconditioning exercise could improve nerve function and reduce infarct size in rats. The underlying mechanism is to regulate the PI3K-AKT pathway through several key genes, promote cerebral angiogenesis, and reduce apoptosis.

Abstract TMP113: RNA Sequencing Reveals Novel Pericytic MiR-15a/16-1 Targets in Post-Stroke Cerebral Angiogenesis

Introduction: Angiogenesis actively contributes to post-stroke functional recovery and improves long-term survival in stroke patients. Previously, we demonstrated that endothelium-targeted deletion of miR-15a/16-1 promotes post-stroke angiogenesis by enhancing classic pro-angiogenic factors and their receptors, and genetic deletion of miR-15a/16-1 in pericytes likewise promotes post-stroke functional recovery by stimulating cerebral angiogenesis. Here, we further investigate the underlying mechanisms and downstream targets of pericytic miR-15a/16-1 in cerebral angiogenesis after ischemic stroke. Methods: Pericyte-miR15a/16-1 conditional knockout mice (Pericyte-miR-15a/16-1 cKO) and wild-type (WT) controls were subjected to 1h MCAO followed by 7d reperfusion. Cerebral microvessels were extracted and subjected to RNA-seq to identify the significant differentially expressed genes (DEGs) that related to miR-15a/16-1 and angiogenesis among experimental groups. TargetScan and miRDB were used to predict potential miR-15a/16-1 downstream targets. Primary mouse brain vascular pericytes (mBVPs) were treated with lentivirus to achieve loss- or gain-of-miR-15a/16-1 function, and subjected to OGD 8h and reperfusion 24h. Pro-angiogenic factors were further verified by dual-luciferase reporter assay, qPCR, and western blotting. Results: RNA-seq analysis showed 289 upregulated genes (P &lt; 0.05 &amp; at least one CPM &gt; 1) and 399 upregulated genes from 100 positive enriched gene sets (GESA) in cerebral microvessels of pericyte-miR-15a/16-1 cKO mice compared to WT controls 7d after MCAO. By comparing to miR-15a/16-1 potential target database from TargetScan and miRDB, 9 angiogenic genes were identified. Further biochemical analysis revealed that Pappa2, Fgf9, Islr, and Ccr2 mRNA and protein expression are significantly increased in cerebral microvessels of cKO mice and mBVPs with loss-of-miR-15a/16-1 function. Furthermore, dual-luciferase assay results verified that loss- or gain-of-miR-15a/16-1 function significantly increased or decreased luciferase reporter activity of these four genes, respectively. Conclusion: Our findings reveal novel pericytic miR-15a/16-1 mediated angiogenic targets in ischemic stroke.

Abstract WP299: Encephalomyosynangiosis Promotes Mature Angiogenesis After Ischemic Stroke

Backgrounds: Current treatments for ischemic stroke patients is ineffective. The brain's inherent ability to self-heal post ischemic stroke is hampered by inadequate blood supply to the affected region. Encephalomyosynangiosis (EMS), a neurosurgical intervention, has proven successful in promoting angiogenesis in patients with Moyamoya disease. We have earlier shown that EMS can increase angiogenesis after stroke in the implanted region, it is not known if those blood vessels are stable or mature. Given that EMS provide balanced growth factors, we therefore hypothesized that EMS enhances stable or mature cerebral angiogenesis in the cortical region surrounding the muscle graft. Methods: Following a 60-minute period of transient middle cerebral artery occlusion (MCAo), mice were assigned randomly to either MCAo alone or MCAo followed by EMS (3-4 hours post MCAo).. Mice were sacrificed at 7, 21, or 70 days post MCAo. A mouse angiogenesis and proteomic profile array was employed to quantify the expression of angiogenic and neuromodulating proteins. Immunohistochemistry was utilized to visualize the bonding of the graft with the brain cortex, changes in vessel density, and pericyte coverage to assess the degree of normal or mature angiogenesis. Results and Conclusions: Preliminary data indicate successful graft implantation and a significantly increased vessel density (P&lt;0.05 compared to the MCAo alone) in proximity to the muscle graft, signifying increased angiogenesis after stroke. EMS-treated mice exhibited an early improvement in sensory motor deficits post-stroke. Analysis of the angiogenesis and proteomic profile array data unveiled a downregulation of antiangiogenic factors (such as interleukin-10 and Fetuin A) and an upregulation of pro-angiogenic cytokines or growth factors (including Fibroblast Growth Factor and C-X-C Motif Chemokine 12) in the EMS group. Co-immunostaining of the endothelial cell marker (Lectin) with the pericyte marker Platelet-Derived Growth Factor Receptor Beta (PDGFRβ) after 70 days of EMS suggested the stability and maturity of new blood vessels. In summary, the EMS procedure appears effective, holding promise for enhanced stable angiogenesis which may help to expedite recovery following ischemic stroke.

Cerebral vascular and blood brain-barrier abnormalities in a mouse model of epilepsy and tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. Invivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.

Stimulation of Cerebral Angiogenesis and Neurogenesis with Transcatheter Intracerebral Laser Photobiomodulation Therapy in Alzheimer’s Disease and Vascular Parkinsonism Aggravated by Dementia

AbstractBackgroundThe research is focused on the stimulation of cerebral angiogenesis and neurogenesis using Transcatheter Intracerebral Laser Photobiomodulation Therapy (PBMT) in patients with dementia in Alzheimer’s disease (AD) and patients with vascular parkinsonism (VP).MethodThe examination of 155 patients included assessment of CDR, The Tomography Dementia Rating scale (TDR), MMSE, cerebral CT, MRI, SG, rheoencephalography (REG) and cerebral MUGA.85 patients were selected, of whom:• 48 (30.97%) people suffering from AD, aged 34‐80 (mean age 67.5), men ‐ 17 (35.42%), women ‐ 31 (54.58%). According to dementia severity, patients were subdivided: preclinical stage (TDR‐0) ‐ 4, mild stage (TDR‐1) ‐ 16, moderately severe stage (TDR‐2) ‐ 21, severe stage (TDR‐3) ‐ 7 patients.• 37 (23.87%) people suffering from VP, aged 58‐80 (mean age 77), men 28 (75.68%), women ‐ 9 (24.32%). 19 (51.35%) had a mild dementia stage (CDR‐1).In hospital conditions, in a specialized cath lab, all patients underwent Transcatheter Intracerebral Laser Photobiomodulation Therapy (PBMT) (wavelength 632.8 nanometers).ResultAll 48 (100%) patients with AD underwent angiogenesis stimulation resulting in cerebral collateral capillary revascularization and closure of atreriovenous shunts.Simultaneously, patients showed a 10‐20% increase in cerebral temporal lobes volume, which indicates cerebral neurogenesis development and tissue regeneration. The process was accompanied by dementia level decrease and cognitive functions restoration. Consequently, patients were transferred to a milder TDR group. Depending on the initial dementia severity, the resulting positive effect was observed from 2 to 15 years.All 37 (100%) patients with VP underwent angiogenesis stimulation resulting in cerebral microcirculation improvement and cerebral involutive changes decrease, which indicates the neurogenesis development and tissue regeneration. The process was accompanied by decreased dementia and cognitive functions restoration. The resulting positive effect was observed over 10 years.ConclusionDespite the differences in AD and VP etiology, Transcatheter Intracerebral Laser Photobiomodulation Therapy (PBMT) is an effective treatment for dementia in these diseases. The method stimulates angiogenesis and improves blood supply to the brain, it stimulates neurogenesis and the restoration of normal tissue structures, which leads to improved mental and cognitive functions. The resulting effect persists for a long time.

Down-Regulation of miRNA-1303 Promotes the Angiogenesis of HUVECs via Targeting THSD7A.

Angiogenesis promotes neurological recovery after acute ischemic stroke (AIS), and microRNAs play crucial roles in cerebral angiogenesis. This study found that Homo sapiens-microRNA-1303(miR-1303) was reduced in blood specimens of AIS patients and human umbilical vein endothelial cells after suffering from oxygen-glucose deprivation/reperfusion. The experiment detected the effect of miR-1303 on angiogenesis by wound healing assay, tube formation assay, and transwell assay. Down-regulation of miRNA-1303 promotes angiogenesis in vitro experiments, while miR-1303 over-expression reverses this effect. Based on bioinformatics analyses and dual-luciferase reporter assay, the thrombospondin type 1 domain containing 7A (THSD7A) was investigated and further validated as the downstream gene of miR-1303. Furthermore, the knockdown of miR-1303 decreased the protein translation and mRNA transcript levels of THSD7A. Our results reveal a novel miR-1303/THSD7A pathway for angiogenesis and further imply that miR-1303 can be a promising biomarker and therapeutic target for AIS.

Cerebral blood flow and cerebrovascular reactivity are modified by maturational stage and exercise training status during youth.

What is the central question of this study? Gonadal hormones modulate cerebrovascular function while insulin-like growth factor 1 (IGF-1) facilitates exercise-mediated cerebral angiogenesis; puberty is a critical period of neurodevelopment alongside elevated gonadal hormone and IGF-1 activity: but whether exercise training across puberty enhances cerebrovascular function is unkown. What is the main finding and its importance? Cerebral blood flow is elevated in endurance trained adolescent males when compared to untrained counterparts. However, cerebrovascular reactivity to hypercapnia is faster in trained vs. untrained children, but not adolescents. Exercise-induced improvements in cerebrovascular function are attainable as early as the first decade of life. Global cerebral blood flow (gCBF) and cerebrovascular reactivity to hypercapnia ( ) are modulated by gonadal hormone activity, while insulin-like growth factor 1 facilitates exercise-mediated cerebral angiogenesis in adults. Whether critical periods of heightened hormonal and neural development during puberty represent an opportunity to further enhance gCBF and is currently unknown. Therefore, we used duplex ultrasound to assess gCBF and in n=128 adolescents characterised as endurance-exercise trained (males: n=30, females: n=36) or untrained (males: n=29, females: n=33). Participants were further categorised as pre- (males: n=35, females: n=33) or post- (males: n=24, females: n=36) peak height velocity (PHV) to determine pubertal or 'maturity' status. Three-factor ANOVA was used to identify main and interaction effects of maturity status, biological sex and training status on gCBF and . Data are reported as group means (SD). Pre-PHV youth demonstrated elevated gCBF and slower mean response times than post-PHV counterparts (both: P≤0.001). gCBF was only elevated in post-PHV trained males when compared to untrained counterparts (634(43) vs. 578(46)mlmin-1 ; P=0.007). However, mean response time was faster in pre- (72(20) vs. 95(29)s; P≤0.001), but not post-PHV (P=0.721) trained youth when compared to untrained counterparts. Cardiorespiratory fitness was associated with gCBF in post-PHV youth (r2 =0.19; P≤0.001) and mean response time in pre-PHV youth (r2 =0.13; P=0.014). Higher cardiorespiratory fitness during adolescence can elevate gCBF while exercise training during childhood primes the development of cerebrovascular function, highlighting the importance of exercise training during the early stages of life in shaping the cerebrovascular phenotype.

Neural stem cell-derived exosomes-loaded adhesive hydrogel controlled-release promotes cerebral angiogenesis and neurological function in ischemic stroke

ObjectiveIschemic stroke has become one of the leading diseases for international death, which brings burden to the economy and society. Exosomes (Exos) derived following neural stem cells (NSCs) stimulation promote neurogenesis and migration of NSCs. However, Exos themselves are easily to be removed in vivo. Our study is to investigate whether adhesive hyaluronic acid (HAD) hydrogel loading NSCs-derived-Exo (HAD-Exo) would promote the recovery of ischemic stroke. MethodsA mouse model of middle cerebral artery occlusion (MCAO) was established. PBS, Exo, HAD, and HAD-Exo groups were independently stereotactically injected in mice, respectively. The modified neurological severity score scale and behaviour tests were used to evaluate neurological improvement. Neuroimagings were used to observe the improvement of cerebral infarct volume and vessels. Immunofluorescence staining was used to verify the expression of vascular and cell proliferation-related proteins. ResultsThe structural and mechanical property of HAD and HAD-Exo were detected. Behavioral results showed that HAD-Exo significantly improved neurological functions, especially motor function. Neuroimagings showed that HAD-Exo significantly promoted infarct volume and angiogenesis. Immunofluorescence staining showed that HAD-Exo significantly promoted the cerebral angiogenesis and anti-inflammation. ConclusionNSCs derived exosomes-loaded adhesive HAD hydrogel controlled-release could promote cerebral angiogenesis and neurological function for ischemic stroke.

Wnt5a-Flt1 activation contributes to preterm altered cerebral angiogenesis after prenatal inflammation.

Intraventricular hemorrhage (IVH) causes morbidity and mortality in preterm infants and prenatal exposure to inflammation contributes to brain injury. Moreover, prenatal exposure to severe inflammation increases the risk of IVH in preterm neonates. The current study investigated whether intrauterine exposure to inflammation affects cerebral angiogenesis and its underlying mechanisms. Wnt5a, flt1, and vascular endothelial growth factor (VEGF)-A levels in cord blood serum (stored in a bio-bank) of the enrolled patients were measured via enzyme-linked immunosorbent assay. A preterm prenatal inflammation exposure model was established in rats by intraperitoneal injection intraperitoneally during pregnancy. Angiogenesis of cerebral tissue was analyzed using immunohistochemistry. Wnt5a, flt1, and VEGF-A expression levels were measured via immunohistochemistry, immunofluorescence, or western blotting. The correlation between Wnt5a and flt1 expression and the cerebral vessel area was also analyzed. The Wnt5a and flt1 levels in the cord blood serum were significantly higher in the amnionitis group than in the non-amnionitis group. The VEGF-A level in the cord blood serum was significantly lower in the amnionitis group. In the rat model, preterm rats in the prenatal inflammation group exhibited increased microglial cell infiltration and decreased vessel area and diameter in the cerebral tissue compared to the control group. Wnt5a was located in microglial cells, and Wnt5a and flt1 expression in brain tissue significantly increased after prenatal lipopolysaccharide (LPS) exposure. VEGF-A expression declined after prenatal LPS exposure. The cerebral vessel area was negatively correlated with Wnt5a and flt1 expression. Disordered cerebral angiogenesis is associated with increased Wnt5a-Flt1 activation in microglial cells after exposure to intrauterine inflammation.

Physiological oxygen conditions enhance the angiogenic properties of extracellular vesicles from human mesenchymal stem cells

BackgroundFollowing an ischemic injury to the brain, the induction of angiogenesis is critical to neurological recovery. The angiogenic benefits of mesenchymal stem cells (MSCs) have been attributed at least in part to the actions of extracellular vesicles (EVs) that they secrete. EVs are membrane-bound vesicles that contain various angiogenic biomolecules capable of eliciting therapeutic responses and are of relevance in cerebral applications due to their ability to cross the blood–brain barrier (BBB). Though MSCs are commonly cultured under oxygen levels present in injected air, when MSCs are cultured under physiologically relevant oxygen conditions (2–9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. There is a need to determine the effects of MSC-EVs in models of cerebral angiogenesis and whether those from MSCs cultured under physiological oxygen provide greater functional effects.MethodsHuman adipose-derived MSCs were grown in clinically relevant serum-free medium and exposed to either headspace oxygen concentrations of 18.4% O2 (normoxic) or 3% O2 (physioxic). EVs were isolated from MSC cultures by differential ultracentrifugation and characterized by their size, concentration of EV specific markers, and their angiogenic protein content. Their functional angiogenic effects were evaluated in vitro by their induction of cerebral microvascular endothelial cell (CMEC) proliferation, tube formation, and angiogenic and tight junction gene expressions.ResultsCompared to normoxic conditions, culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance.ConclusionsEVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function.

Possibility of stimulation of cerebral angiogenesis and neurogenesis with transcatheter intracerebral laser photobiomodulation therapy during dementia in patients with Alzheimer’s disease and in patients with distal cerebral atherosclerosis

AbstractBackgroundThe research is dedicated to stimulation of cerebral angiogenesis and neurogenesis with Transcatheter Intracerebral Laser Photobiomodulation Therapy (PBMT) in patients with dementia during Alzheimer’s disease (AD) and patients with distal forms of cerebral atherosclerosis.Materials &amp; Methods350 patients with dementia were examined.We carried out determination of CDR, Tomography Dementia Rating scale (TDR), MMSE, cerebral CT, MRI, SG, rheoencephalography (REG), cerebral MUGA.48 (13.71%) patients with AD aged 34‐80 (mean age 67.5) were selected: 17 men (35.42%), 31 women (54.58%). According to their dementia severity, the patients were subdivided: preclinical stage (TDR‐0) ‐ 4, mild stage (TDR‐1) ‐ 16, moderately severe stage (TDR‐2) ‐ 21, severe stage (TDR‐3) ‐ 7.302 (86.29%) patients with distal cerebral atherosclerosis aged 58‐81 (mean age 78) were selected: 226 men (74.83%), 76 women (25.17%). According to their dementia severity, the patients were subdivided: mild stage (СDR‐1) ‐ 284 (94.04%), moderately severe stage (СDR‐2) ‐ 18 (5.96%).At hospital, all the patients underwent PBMT.ResultsAll 48 (100%) patients with AD showed improved cerebral microcirculation and 10‐20% increase in the volume of cerebral temporal lobes, which indicates cerebral neurogenesis development and tissue regeneration. The process was accompanied by a decreased level of dementia and restoration of cognitive functions. Consequently, the patients were transferred to milder TDR groups. Depending on the initial dementia severity, the resulting positive effect was observed during 2‐10 years.All 302 (100%) patients with distal cerebral atherosclerosis showed improved cerebral microcirculation and declining cerebral involutional changes, which indicates neurogenesis development and tissue regeneration. The process was accompanied by a decreased dementia level and cognitive functions restoration. The resulting positive effect was observed for over 10 years.ConclusionsDespite differences in the etiology of AD and distal cerebral atherosclerosis, PBMT is an effective treatment for dementia in these diseases. The method improves the blood supply of the brain; it stimulates neurogenesis and tissue structures restoration, which leads to an improvement in mental and cognitive functions. The resulting effect persists for a long time.

Danshensu Enhances Cerebral Angiogenesis in Mice by Regulating the PI3K/Akt/Mtor/VEGF Signaling Axis.

Cerebral infraction seriously affects the life quality of patients. Danshensu has been reported to exhibit anti-inflammatory and vascular protective effects. However, the therapeutic function of Danshensu in cerebral vascular injury is still unclear. Middle cerebral artery occlusion (MCAO) was used to construct the cerebral infraction model. Wound healing and tube formation assays were used to evaluate angiogenesis in vitro. Western blot assay was used to evaluate the activation of the PI3K/Akt/mTOR signaling pathway. The laser Doppler scanner was used to measure the regional cerebral blood flow (rCBF) in the area around the infarction, and the adhesion removal test was used to measure the sensorimotor function. The Modified Neurological Severity Score was performed to evaluate the cognitive functions of mice. Danshensu promoted the proliferation of bEnd.3 cells and angiogenesis in vitro. Danshensu upregulated the expression of VEGF through PI3K/Akt/mTOR signaling pathway in bEnd.3 cells. Danshensu improved rCBF restoration and attenuated the behavioral deficits in mice post-MCAO/R. Danshensu enhances angiogenesis through the PI3K/Akt/mTOR/VEGF signaling pathway in a mouse model of cerebral ischemic injury.

Long-term intermittent fasting improves neurological function by promoting angiogenesis after cerebral ischemia via growth differentiation factor 11 signaling activation.

Intermittent fasting (IF), an alternative to caloric restriction, is a form of time restricted eating. IF conditioning has been suggested to have neuroprotective effects and potential long-term brain health benefits. But the mechanism underlying remains unclear. The present study focused on the cerebral angiogenesis effect of IF on ischemic rats. Using a rat middle cerebral artery occlusion model, we assessed neurological outcomes and various vascular parameters such as microvessel density (MVD), regional cerebral blood flow (rCBF), proliferation of endothelial cells (ECs), and functional vessels in the peri-infarct area. IF conditioning ameliorated the modified neurological severity score and adhesive removal test, increased MVD, and activated growth differentiation factor 11 (GDF11)/activin-like kinase 5 (ALK5) pathways in a time-dependent manner. In addition, long-term IF conditioning stimulated proliferation of ECs, promoted rCBF, and upregulated the total vessel surface area as well as the number of microvessel branch points through GDF11/ALK5 pathways. These data suggest that long-term IF conditioning improves neurological outcomes after cerebral ischemia, and that this positive effect is mediated partly by angiogenesis in the peri-infarct area and improvement of functional perfusion microvessels in part by activating the GDF11/ALK5 signaling pathway.

Elabela-APJ axis mediates angiogenesis via YAP/TAZ pathway in cerebral ischemia/reperfusion injury

Angiogenesis helps to improve neurological recovery by repairing damaged brain tissue and restoring cerebral blood flow (CBF). The role of the Elabela (ELA)-Apelin receptor (APJ) system in angiogenesis has gained much attention. We aimed to investigate the function of endothelial ELA on post-ischemic cerebral angiogenesis. Here, we demonstrated that the endothelial ELA expression was upregulated in the ischemic brain and treatment with ELA-32 mitigated brain injury and enhanced the restoration of CBF and newly formed functional vessels following cerebral ischemia/reperfusion (I/R) injury. Furthermore, ELA-32 incubation potentiated proliferation, migration, and tube formation abilities of the mouse brain endothelial cells (bEnd.3 cells) under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. RNA sequencing analysis indicated that ELA-32 incubation had a role in the Hippo signaling pathway, and improved angiogenesis-related gene expression in OGD/R-exposed bEnd.3 cells. Mechanistically, we depicted that ELA could bind to APJ and subsequently activate YAP/TAZ signaling pathway. Silence of APJ or pharmacological blockade of YAP abolished the pro-angiogenesis effects of ELA-32. Together, these findings highlight the ELA-APJ axis as a potential therapeutic strategy for ischemic stroke by showing how activation of this pathway promotes post-stroke angiogenesis.

Mechanism of neocryptotanshinone in protecting against cerebral ischemic injury: By suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis

AimThis study explored the protective function and mechanism of neocryptotanshinone (NEO) on cerebral ischemia. MethodsLipopolysaccharide/γ-interferon(LPS/IFN-γ)was employed to mimic the polarization of mouse microglial cells BV2. After NEO treatment, the M1 polarization level of BV2 cells was identified using flow cytometry (FCM), fluorescent cell staining and enzyme linked immunosorbent assay(ELISA). Moreover, the mouse endothelial cells bEnd.3 were applied to be the study objects, which were intervened with NEO under the hypoxic condition. Thereafter, based on in-vitro tubule formation assay and fluorescence staining, the in-vitro tubule formation ability of bEnd.3 cells was detected. By adopting middle cerebral artery occlusion(MCAO) method, we constructed the mouse model of cerebral ischemia. After NEO intervention, the pathological changes of brain tissues were identified, while CD34 expression was measured by immunohistochemical (IHC) staining, nerve injury was detected by Nissl staining, and the changes in neurological behaviors of mice were also detected. ResultsOur results showed that NEO suppressed M1 polarization of BV2 cells, which exerted its effect through suppressing NF-κB and STAT3 signals, thereby decreasing the levels of iNOS, CD11b and inflammatory factors. NEO stimulated tubule formation in bEnd.3 cells based on the hypoxic situation, which exerted its effect through activating the Vascularendothelial growth factor-Vascular Endothelial Growth Factor Receptor 2-Notch homolog 1(VFGF-VEGFR2-Notch1) signal. Furthermore, NEO suppressed cerebral ischemia in mice and lowered the ischemic penumbra. NEO also improved the neurological behaviors of mice, increased the CD34 levels and decreased the expression of inflammatory factors. ConclusionNEO has well protective effect against cerebral ischemia, and its mechanisms are related to suppressing M1 polarization of microglial cells and promoting cerebral angiogenesis, which are the mechanisms of NEO in treating ischemic encephalopathy.