Endothelial Cell Proliferation In Response Research Articles

Abstract 124: Renin Cell Identity And Blood Pressure Homeostasis: Exploring The Role Of Endothelial Cell Cross-talk

Our previous studies using an Aortic Coarctation (AoCo) mouse model identified that renin-expressing juxta-glomerular (JG) cells respond to changes in perfusion pressure and Lamin A/C as a component of the renin cell baroreceptor. However, as the mechanisms for transmitting the extracellular pressure to the JG cells are unclear, we hypothesized that the endothelial cells (ECs) act as transducers of signals to the JG cells. Therefore in the current study, we aim 1) to identify the concurrent transcriptomic and epigenomic changes in JG cells and ECs in response to the changes in the perfusion pressure by single-nuclei multiomics approach 2) to define the contribution of EC-specific Lamin A/C (Lmna) in the EC-JG-cells cross-talk. We performed the AoCo surgeries on 3 months (M) old VE-Cad-Cre;Foxd1-Cre;mTmG mice where GFP is expressed simultaneously in Foxd1 and EC-lineage cells. We compared the single-cell gene expression and chromatin accessibility between the left (LK) and the right kidneys (RK) at 72h post-surgeries. Our study revealed: 1) JG cells increased significantly in the LK along with renin gene expression 2) ECs significantly increased by 10.73% in LK with a higher expression for genes promoting EC proliferation, morphogenesis, and immune response 3) JGs of the LK showed enrichment for the motifs Mbd1, Smad1, Smad5 indicating epigenetic changes promoting transcriptional regulation and TgfB1 signaling. Enrichment of Mybl1, Myb, and E2f2 motifs in the ECs of the LK suggests positive regulation of EC proliferation in response to low blood pressure (BP). Furthermore, deletion of the Lmna gene in the EC-lineage affected the BP responses in 4M old Lmna fl/fl ;VE-Cad.Cre mice. The difference between the carotid and femoral mean BP (MBP) measurements was more prominent in the wild type (WT) compared to the mutants at 72h post-AoCo [WT (n=7), Carotid MBP: 69.16±2.24 Femoral MBP: 63.28±2.33, P<0.00001 Mutants (n=4); Carotid MBP: 75.98±3.06 Femoral MBP: 69.75±3.10, P<0.05)]. Our current study shows that ECs undergo transcriptional and epigenetic alterations in response to changes in perfusion pressure. Deletion of Lmna in ECs reduces the differences in the BP between LK and RK, suggesting that it is important for sensing the changes in extracellular pressure.

Mechanoresponsive Smad5 Enhances MiR-487a Processing to Promote Vascular Endothelial Proliferation in Response to Disturbed Flow.

MicroRNAs (miRs) and bone morphogenetic protein receptor–specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3′UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow–associated vascular disorders resulting from atherosclerosis.

Mechanosensitive Smad5 Enhances MiR‐487a Processing to Regulate Vascular Endothelial Cell Proliferation in Response to Disturbed Flow

MicroRNAs (miRs) and bone morphogenetic protein receptor‐specific Smads are mechano‐responsive molecules that play vital roles in modulating endothelial cell (EC) functions in the face of blood flow. However, the roles of interplay between these molecules in modulating EC functions under different flows remain unclear. We elucidated the roles of interplay between miRs and Smad5 in modulating EC proliferation in response to different flows. Microarray and quantitative RT‐PCR showed that disturbed flow with oscillatory shear stress (OS, 0.5±4 dynes/cm2) up‐regulates EC miR‐487a in comparison to static controls and pulsatile shear stress (12±4 dynes/cm2). MiR‐487a was up‐regulated in ECs in the inner curvature (OS region) of rat aortic arch and diseased human coronary arteries. MiR‐487a expression was promoted by nuclear phospho‐Smad5, which bound to primary‐miR‐487a to facilitate miR‐487a processing. Algorithm prediction, luciferase reporter, and argonaute 2‐immunoprecipitation assays showed that miR‐487a binds to 3’UTR of CREB binding protein (CBP) and p53. Knockdown or overexpression of miR‐487a decreased and increased, respectively, phospho‐Rb and cyclin A expressions through CBP and p53. BrdU incorporation assay showed that miR‐487a enhances EC proliferation under OSS in vitro and in disturbed flow regions of experimentally‐stenosed rat abdominal aorta in vivo. In summary, disturbed flow with OSS induces EC expression of miR‐487a through its enhanced processing by activated‐Smad5. MiR‐487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR‐487 is an important molecular target for intervention against disturbed flow‐associated vascular diseases resulting from atherosclerosis.

Muscle‐derived SDF‐1α/CXCL12 modulates endothelial cell proliferation but is not required for exercise training‐induced angiogenesis

Chronic elevations in contractile activity, as observed with exercise training, increase capillarity density in skeletal muscle. Here, we demonstrate that the stromal cell‐derived factor‐1 alpha (SDF‐1α/CXCL12) is the most abundantly expressed angiogenesis‐associated chemokine in capillary‐rich oxidative soleus and exercise‐trained plantaris muscles. CXCL12 is also increased in muscle‐specific peroxisome proliferator‐activated receptor γ coactivator 1α transgenic mice (PGC‐1α mTg) when compared to wild‐type littermates. Additionally, CXCL12 is increased in C2C12 myotubes by cyclic mechanical stretch. Furthermore, exposure of human umbilical vein endothelial cells (HUVEC) to conditioned medium from cyclically stretched C2C12 myotubes enhanced cell proliferation. Such an effect was specific to CXCL12 signaling because co‐treatment with AMD3100, an antagonist of the CXCL12 receptor CXCR4, disrupted the proliferative response. To gain further insight into a potential role for CXCL12 in modulating chronic contractile activity‐induced angiogenesis in vivo, we studied muscle‐specific CXCL12 knockout mice (CXCL12 mKO). Interestingly, our results demonstrate that these mice have normal baseline capillary density and preserved angiogenesis when exercise‐trained. These findings are consistent with a muscle‐derived CXCL12‐dependent modulatory role of endothelial cell proliferation in response to increased contractile activity. However, muscle‐derived CXCL12 is not required for exercise training‐induced angiogenesis in skeletal muscle.Support or Funding InformationThis study was supported by Grant‐in‐Aid for Challenging Exploratory Research (16K13019) and Grant‐in‐Aid for Scientific Research (B) (15H03080) (18H03153) to Mitsuharu Okutsu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spatiotemporal release of VEGF from biodegradable polylactic-co-glycolic acid microspheres induces angiogenesis in chick chorionic allantoic membrane assay

While vascular endothelial growth factor (VEGF) is an acknowledged potent pro-angiogenic agent there is a need to deliver it at an appropriate concentration for several days to achieve angiogenesis. The aim of this study was to produce microspheres of biodegradable polylactic-co-glycolic acid (PLGA) tailored to achieve sustained release of VEGF at an appropriate concentration over seven days, avoiding excessive unregulated release of VEGF that has been associated with the formation of leaky blood vessels. Several formulations were examined to produce microspheres loaded with both human serum albumin (HSA) and VEGF to achieve release of VEGF between 3 and 10 ng per ml for seven days to match the therapeutic window desired for angiogenesis. In vitro experiments showed an increase in endothelial cell proliferation in response to microspheres bearing VEGF. Similarly, when microspheres containing VEGF were added to the chorionic membrane of fertilised chicken eggs, there was an increase in the development of blood vessels over seven days in response, which was significant for microspheres bearing VEGF and HSA, but not VEGF alone. There was an increase in both blood vessel density and branching – both signs of proangiogenic activity. Further, there was clearly migration of cells to the VEGF loaded microspheres. In summary, we describe the development of an injectable delivery vehicle to achieve spatiotemporal release of physiologically relevant levels of VEGF for several days and demonstrate the angiogenic response to this. We propose that such a treatment vehicle would be suitable for the treatment of ischemic tissue or wounds.

In vitro endothelial cell proliferation assay reveals distinct levels of proangiogenic cytokines characterizing sera of healthy subjects and of patients with heart failure.

Although myocardial angiogenesis is thought to play an important role in heart failure (HF), the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52 ± 7.6 years; n = 46) healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29 ± 8.6 years; n = 20). The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients' sera, we observed that a subset of sera (n = 11) promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n = 18). Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P < 0.05) differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48h) PASMC were stimulated with IL-1β alone or with NO donor, L-arginine and/or NO synthase inhibitor L-NAME for 4 and 24h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24h. VEGF release was inhibited by L-NAME (531.8±52pg/ml), but restored and further elevated by L-arginine (1,529±287pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by L-NAME (1.1-fold) and augmented by L-arginine (3.8-fold) at 4h. Restoration of a NO pathway by L-arginine in L-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [(3)H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.

BMP receptor-integrin interaction mediates responses of vascular endothelial Smad1/5 and proliferation to disturbed flow

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress. Our previous study demonstrated that disturbed flow with low and oscillatory shear stress (OSS) induces bone morphogenetic protein receptor (BMPR)-specific Smad1/5 activation in ECs, but the underlying mechanisms and the in vivo functional role of Smad1/5 remain unclear. Here we elucidated the molecular mechanisms by which OSS activates EC Smad1/5 and its in vivo functional role. Lentiviral Smad5-specific short hairpin RNA (Lenti-shSmad5) was constructed and intra-arterially injected into the lumen of stenosed abdominal aorta in bromodeoxyuridine-infused rats. Co-immunoprecipitation and in situ proximity ligation assays were performed on ECs exposed to OSS (0.5±4 dynes/cm(2) ) in a parallel-plate flow chamber to investigate BMPR-integrin interactions and their regulatory role in OSS-activation of EC Smad1/5. Intra-arterial administration of Lenti-shSmad5 inhibited bromodeoxyuridine uptake of ECs at post-stenotic sites, where disturbed flow with OSS occurs. OSS induced sustained BMPRIB-αv β3 integrin association in ECs, which was mediated by the intracytoplasmic kinase domain of BMPRII and subsequently activated the Shc/focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) cascade, leading to Smad1/5 activation. This OSS-activation of Smad1/5 induced its association with and activation of runt-related transcription factor-2 (Runx2), leading to activations of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K), a pathway critical for EC proliferation in response to OSS. Oscillatory shear stress induces synergistic interactions between specific BMPRs and integrin to activate Smad1/5 through the Shc/FAK/ERK pathway, which leads to the activation of the Runx2/mTOR/p70S6K pathway to promote EC proliferation.

Fatty Acid Binding Protein 4 Regulates VEGF-Induced Airway Angiogenesis and Inflammation in a Transgenic Mouse Model: Implications for Asthma

Neovascularization of the airways occurs in several inflammatory lung diseases, including asthma. Vascular endothelial growth factor (VEGF) plays an important role in vascular remodeling in the asthmatic airways. Fatty acid binding protein 4 (FABP4 or aP2) is an intracellular lipid chaperone that is induced by VEGF in endothelial cells. FABP4 exhibits a proangiogenic function invitro, but whether it plays a role in modulation of angiogenesis invivo is not known. We hypothesized that FABP4 promotes VEGF-induced airway angiogenesis and investigated this hypothesis with the use of a transgenic mouse model with inducible overexpression of VEGF165 under a CC10 promoter [VEGF-TG (transgenic) mice]. We found a significant increase in FABP4 mRNA levels and density of FABP4-expressing vascular endothelial cells in mouse airways with VEGF overexpression. FABP4(-/-) mouse airways showed a significant decrease in neovessel formation and endothelial cell proliferation in response to VEGF overexpression. These alterations in airway vasculature were accompanied by attenuated expression of proinflammatory mediators. Furthermore, VEGF-TG/FABP4(-/-) mice showed markedly decreased expression of endothelial nitric oxide synthase, a well-known mediator of VEGF-induced responses, compared with VEGF-TG mice. Finally, the density of FABP4-immunoreactive vessels in endobronchial biopsy specimens was significantly higher in patients with asthma than in control subjects. Taken together, these data unravel FABP4 as a potential target of pathologic airway remodeling in asthma.

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.

Mechanical Activation of Smad, A Novel Regulator for Endothelial Cell Proliferation Induced by Disturbed Flow

Atherosclerotic lesions develop at arterial branches and curvatures, where the local flow is disturbed with low and oscillatory shear stress (OSS). We investigated the role of OSS in the endothelial cell (EC) activations of Smad1/5 and their regulation in OSS‐induced EC proliferation. OSS (0.5±4 dynes/cm2) to human umbilical vein ECs induced sustained phosphorylations of Smad1/5. Smad1/5 activations were found only in the selective patches of ECs overlying foam cell lesions in human coronary arteries. These shear‐activations of Smad1/5 were not inhibited by pre‐treating ECs with Noggin, a specific antagonist of bone morphogenic proteins. Transfecting ECs with specific siRNA of BMPRII, BMPRIB, and αv and β3 integrins inhibited OSS‐induced Smad1/5 phosphorylations. OSS induced sustained increases in the association of αvβ3 integrin with BMPRIB, which was inhibited by BMPRII‐specific siRNA. OSS induced the activations of focal adhesion kinase/Shc/extracellular signal‐regulated kinase, which are required for OSS‐induced Smad1/5 phosphorylations. OSS induced increased expression of cyclin A and decreased expressions of p21CIP1 and p27KIP1 and hence proliferation in ECs. Transfecting ECs with Smad1/5‐specific siRNA inhibited these OSS‐induced responses in ECs. Our findings provide a molecular basis for the role of Smad1/5 in regulating EC proliferation in response to OSS.

PKCα-MAPK/ERK-phospholipase A 2 signaling is required for human melanoma-enhanced brain endothelial cell proliferation and motility

The largely undefined signal transduction mechanisms and cross-talk between human melanoma cell (HMC) lines and brain endothelial cells (ECs) involved in tumor cell interaction and adhesion were investigated. In immortalized rat brain GP8.3 EC cultures, conditioned media (CM) prepared from SK-MEL28 and OCM-1 melanoma cells significantly enhanced arachidonic acid release, cytosolic phospholipase A 2 (cPLA 2) and Ca +-independent phospholipase A 2 (iPLA 2) specific activities, and cell growth by 24 h. Inhibitors such as wortmannin and LY294002 (vs. PI3 kinase activity), AACOCF 3, (vs. cPLA 2 and iPLA 2), PD98059 (vs. ERK1/2 activity) and NS-398 (vs. cyclooxygenase-2 activity, COX-2) were all able to block cell proliferation and motility determined using a scratch wound healing assay in melanoma CMs-stimulated EC monolayers. These media also support the enhanced cell proliferation of primary ECs derived from rat brain (BBEC). Electroporation of anti-cPLA 2 antibody into ECs markedly inhibited the EC proliferation in response to CMs. With both CMs, phosphorylation of cPLA 2, PKCα, ERK1/2, protein and mRNA expression of cPLA 2 and iPLA 2, and COX-2 protein expression were significantly stimulated after 24 h coincubation, and attenuated by specific inhibitors. By confocal microscopy, activation of cPLA 2, ERK1/2, PKCα and COX-2 in perinuclear and membrane regions of ECs grown in CM-stimulated cultures were clearly observed. Thus MEK-PKCα-ERK1/2 and PI3-K/Akt survival pathways are activated in EC cultures during the interaction with CM from both melanoma cell lines, providing new insight in understanding EC metabolism and signaling. These pathways represent potential therapeutic targets to inhibit or enhance tumor angiogenesis.

Endostatin and anastellin inhibit distinct aspects of the angiogenic process

BackgroundEndostatin and anastellin, fragments of collagen type XVIII and fibronectin, respectively, belong to a family of endogenous inhibitors of angiogenesis which inhibit tumor growth and metastasis in a number of mouse models of human cancer. The mechanism of action of these inhibitors is not well understood, but they have great potential usefulness as non-toxic long-term therapy for cancer treatment.MethodsIn this study, we compare the anti-angiogenic properties of endostatin and anastellin using cell proliferation and transwell migration assays.ResultsAnastellin but not endostatin completely inhibited human dermal microvessel endothelial cell proliferation in response to serum stimulation. Both anastellin and endostatin additively inhibited endothelial cell migration in response to VEGF. Anastellin but not endostatin lowered basal levels of active ERK.ConclusionThese data indicate that anastellin and endostatin exert their anti-angiogenic effects by modulating distinct steps in the angiogenic pathway and suggest that matrix-derived inhibitors of angiogenesis may exhibit higher efficacy when used in combination.

Proangiogenic Effects of α1‐Adrenergic Receptor Blockade

We studied the role of α1-adrenergic receptors (AR) in ischemia-induced neoangiogenesis (NeoA) in vivo in 12-week-old rats in which the right common femoral artery was removed and in vitro in cultured rat endothelial cells (EC). We investigated the effects of 14 days’ administration of the α1-AR antagonist doxazosin (DOXA, 0.06 mg/kg/d, n=16) or saline (CTRL, n=6) by means of peritoneum-implanted osmotic minipumps. Laser Doppler analysis and digital angiography TIMI Frame Count showed an improvement in perfusion of the ischemic hind limb of the DOXA vs CTRL group. Similarly, dyed microsphere perfusion and histologic evaluation confirmed the beneficial effects of DOXA on NeoA. In vitro, we verified by RT-PCR the expression of α1-AR in EC. DOXA (10−7M; 24 h) increased EC proliferation in response to fetal bovine serum (FBS, 5%) and enhanced FBS-induced ERK and Rb activation. Our findings support a beneficial role for α1-AR blockade in ischemia-induced NeoA and on EC proliferation. Research support from Pfizer Inc.

Hypoxia-Induced Endothelial Proliferation Requires Both mTORC1 and mTORC2

A central regulator of cell growth that has been implicated in responses to stress such as hypoxia is mTOR (mammalian Target Of Rapamycin). We have shown previously that mTOR is required for angiogenesis in vitro and endothelial cell proliferation in response to hypoxia. Here we have investigated mTOR-associated signaling components under hypoxia and their effects on cell proliferation in rat aortic endothelial cells (RAECs). Hypoxia (1% O(2)) rapidly (>30 minutes) and in a concentration-dependent manner promoted rapamycin-sensitive and sustained phosphorylation of mTOR-Ser2448 followed by nuclear translocation in RAECs. Similarly, hypoxia induced phosphorylation of the mTORC2 substrate Akt-Ser473 (3 to 6 hours at 1% O(2)) and a brief phosphorylation peak of the mTORC1 substrate S6 kinase-Thr389 (10 to 60 minutes). Phosphorylation of Akt was inhibited by mTOR knockdown and partially with rapamycin. mTOR knockdown, rapamycin, or Akt inhibition specifically and significantly inhibited proliferation of serum-starved RAECs under hypoxia (P<0.05; n> or =4). Similarly, hypoxia induced Akt-dependent and rapamycin-sensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1alpha knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (P<0.001; n=7), and mTORC1 inhibition by raptor silencing, partially (P<0.05; n=7), inhibited hypoxia-induced RAEC proliferation. Thus, mTOR mediates an early response to hypoxia via mTORC1 followed by mTORC2, promoting endothelial proliferation mainly via Akt signaling. mTORC1 and especially mTORC2 might therefore play important roles in diseases associated with hypoxia and altered angiogenesis.

Region Specific Hypothalamic Neuronal Activation and Endothelial Cell Proliferation in Response to Electroconvulsive Seizures

Major depression is often associated with disturbances in basal biological functions regulated by the hypothalamus. Electroconvulsive therapy (ECT), an efficient anti-depressant treatment, alters the activity of hypothalamic neurons. We have previously shown an increased proliferation of endothelial cells in specific areas of the rat hippocampus in response to electroconvulsive seizure (ECS) treatment, an animal model for ECT. Here we examine the effect of ECS treatment on neuronal activation and endothelial cell proliferation in mid-hypothalamus. Rats received one daily ECS treatment for 5 days and cell proliferation was detected by bromodeoxyuridine (BrdU). The number of cells double-labeled for BrdU and the endothelial cell marker rat endothelial cell antigen-1 was determined. Neuronal activation in response to acute ECS treatment was detected as c-Fos immunoreactivity in an additional experiment. We demonstrate a correlating pattern of increases in neuronal activation and increased endothelial cell proliferation in the paraventricular nucleus, the supraoptic nucleus, and the ventromedial nucleus of the hypothalamus after ECS treatment. Hypothalamic areas with the largest increase in neuronal activation after ECS treatment exhibit increased endothelial cell proliferation. We suggest that similar angiogenic responses to ECT might counteract hypothalamic dysfunction in depressive disorder.

Expression and regulation of neuropilins and VEGF receptors byTNF-α in human endothelial cells

Recently, neuropilins (NRPs) were identified as new VEGF receptors (VEGFRs) and VEGFR-3 was revealed to be implicated in angiogenesis besides in lymphangiogenesis. However, quantitative expression and regulation of NRPs and VEGFRs remain unclear. The objective of this study was to compare the expression of NRPs and VEGFRs and investigate their regulation by tumor necrosis factor (TNF-alpha) in human endothelial cells (ECs) as well as their roles in VEGF(165)-induced EC proliferation. Human umbilical vein ECs (HUVECs) were treated with TNF-alpha (2 ng/ml) or PBS for 24 h. The mRNA and protein levels of NRP-1, NRP-2, VEGFR-1, VEGFR-2, and VEGFR-3 were semiquantitatively determined by real-time PCR and Western blot, respectively. Real-time PCR data were presented as the difference of reaction cycle thresholds (Ct) between beta-actin and each of the genes of interest (2(-DeltaCt)). EC proliferation in response to VEGF(165) (10 ng/ml) with or without anti-VEGFR-2 neutralization antibody pretreatment was analyzed by [(3)H]thymidine incorporation. In PBS-treated HUVECs, mRNA levels of NRPs and VEGFRs were NRP-1 (0.013), NRP-2 (0.007), VEGFR-2 (0.006), VEGFR-1 (0.0024), and VEGFR-3 (0.0009). After TNF-alpha treatment, mRNA levels of VEGFR-2, VEGFR-3, and NRP-1 were significantly reduced by 72, 65, and 53%, respectively (P < 0.05). The protein expression of all NRPs and VEGFRs were also detected by Western blot. TNF-alpha significantly reduced protein levels of VEGFR-2, VEGFR-3, and NRP-1 by 59, 35, and 22%, respectively. However, both mRNA and protein levels of VEGFR-1 and NRP-2 were not affected by TNF-alpha. Furthermore, TNF-alpha treatment significantly reduced EC proliferation in response to VEGF(165) by 67%. After blocking VEGFR-2 with neutralization antibody, TNF-alpha treatment elicited a 30% reduction of EC proliferation in response to VEGF(165). These data demonstrate that HUVECs express higher mRNA levels of NRP-1 and NRP-2 than those of VEGFRs, and TNF-alpha treatment significantly decreases the expression of VEGFR-2, VEGFR-3, and NRP-1, which may be responsible for TNF-alpha-induced reduction of EC proliferation in response to VEGF(165).

RhoA activation mediates phosphatidylinositol 3-kinase-dependent proliferation of human vascular endothelial cells: an alloimmune mechanism of chronic allograft nephropathy.

HLA class I ligation on graft endothelial cells (EC) has been shown to promote graft arteriosclerosis and chronic allograft nephropathy. This study investigated transcriptional and functional changes mediated by anti-HLA antibodies (Ab), developed by transplant recipient, on vascular renal EC. For mimicking interactions that occur between alloantibodies and graft endothelium, HLA-typed primary cultures of human EC were incubated in vitro in the presence of monomorphic or polymorphic anti-HLA class I Ab. Gene expression analysis identified the upregulation of several molecules involved in cell signaling and proliferation, including the GTP-binding protein RhoA. It was demonstrated further that HLA class I ligation on EC induced a rapid translocation of RhoA to the cell membrane associated with F-actin stress fiber formation and cytoskeleton reorganization. Western blot analysis showed that anti-HLA class I Ab induced, in addition to RhoA, the activation of phosphatidylinositol 3-kinase, reflected by the phosphorylation of Akt (Ser473) and GSK3beta (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 micromol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth.

Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4.

Endothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21 waf1/cip1 and p27 kip1 expressions; 3) EHNA (20 micro M), a selective PDE2 inhibitor, RP73401 (10 micro M), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27 kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27 kip1. 6) EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21 waf1/cip1 expressions; 7) Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects.

Notch Activation during Endothelial Cell Network Formation in Vitro Targets the Basic HLH Transcription Factor HESR-1 and Downregulates VEGFR-2/KDR Expression

Angiogenesis is essential for normal homeostasis, wound healing, and tumor growth and involves a switch in endothelial cell (EC) phenotype from quiescence to migration, proliferation and network formation, and back to quiescence. The notch signaling pathway is critically involved in cell fate decisions during development, and mice deficient in several notch/notch ligand genes have vascular phenotypes. Here we show that notch signaling is activated during EC capillary-like network formation in vitro and that EC express transcripts for notch 1, notch 4, the notch ligand delta 4, and the putative notch processing enzymes ADAM-10 and presenilin. Expression of dominant negative notch blocks network formation; however, constitutively active notch (NICD) does not induce morphologic changes. Furthermore, both EC network formation and expression of activated notch 1 or notch 4 induce expression of the bHLH transcription factor HESR-1 and downregulate the known HESR-1 target VEGFR-2 (KDR). Notch-mediated reduction in VEGFR-2 expression results in decreased EC proliferation in response to VEGF but not bFGF. These data suggest that HESR-1 may be involved in the phenotypic changes that characterize the progression from EC proliferation and migration to network formation and quiescence.