Activation Of Vascular Endothelial Growth Factor Receptor Research Articles

7410 Bevacizumab Induced Hypothyroidism

Abstract Disclosure: A.L. McKenna: None. S. Thota-Kammili: None. K. Grennan: None. S. Rao: None. It is known that thyroid dysfunction is an adverse effect of tyrosine kinase inhibitors (TKIs) via inhibition of the vascular endothelial growth factor receptor (VEGFR). The VEGF signaling pathway is important in regulating angiogenesis in tumors and normal tissue. As a highly vascularized gland, it is expected the thyroid may be compromised because of VEGFR inhibitors. Proposed mechanisms of thyroid dysfunction include capillary regression and constriction leading to reduced vascular perfusion to the gland. Bevacizumab is a monoclonal antibody that targets VEGF and inhibits activation of VEGFR, reducing angiogenesis; interestingly, this is not commonly reported in the literature. Here we present a case where bevacizumab altered thyroid function in a patient with pre-existing primary hypothyroidism. A 60-year-old woman presented with worsening hypothyroidism. She reported a history of nonautoimmune primary hypothyroidism diagnosed in her early 20s; she still had an intact thyroid with no history of head/neck radiation. In the months leading to the presentation, she had been receiving maintenance chemotherapy with 5 fluorouracil and bevacizumab every 2 weeks for metastatic duodenal carcinoma. Prior to starting chemotherapy, she had been on a steady dose of 112 mcg of levothyroxine (LT4) daily. After 6 months on chemotherapy, she reported symptoms of fatigue and hair loss. Workup revealed an elevated TSH of 41 mIU/L (reference 0.3-4.2 mIU/L). She denied any significant changes in weight or malabsorption issues. Aside from the chemotherapy, no other new medications were introduced. Her LT4 dose was increased to 137 mcg daily, and 3 months later, there was improvement in TSH to 6.5 mIU/L as well as her symptoms. At this point, bevacizumab was briefly on hold for 2 months due to an upcoming procedure, hence there was no change to LT4 to avoid over correction. Her TSH normalized and was trending lower while bevacizumab was on hold. Once bevacizumab resumed, LT4 was preemptively increased to 150 mcg daily. Four months after resuming bevacizumab, her TSH remains within the normal reference range on 150 mcg LT4. It is hypothesized that this VEGF blocker led to worsening of her underlying hypothyroidism.Given the shared down regulation in the VEGF signaling pathway, it would be expected that TKIs and monoclonal antibodies against VEGF would share similar adverse reactions to the thyroid. However, to our knowledge, there have been limited reports of bevacizumab induced thyroid dysfunction in the literature. Explanations to this may include the more targeted VEGF inhibition with bevacizumab versus the inhibition of multiple kinase receptors from the TKIs. Further research into this will be needed. This case demonstrates that bevacizumab can impact thyroid function and thyroid labs may need to be monitored at the onset and during VEGF inhibitor therapies. Presentation: 6/3/2024

Clinical course and vascular endothelial growth factor signaling system expression in maxillary angiosarcoma: A case report.

Maxillary angiosarcoma, an aggressive tumor derived from vascular endothelial cells, is very rare. Recently, antivascular endothelial growth factor (VEGF) therapies have attracted considerable attention. We describe the clinical course of a patient with maxillary angiosarcoma and discuss the expression of VEGF signaling molecules assessed via immunohistological analysis. An 81-year-old man presented with an aggressive tumor in the left maxillary sinus. Biopsy revealed atypical nuclear cell proliferation, and the tumor was suspected to be a sarcoma. The maxillary malignancy was treated using a multidisciplinary approach with a combination of surgery, radiotherapy, and regional chemotherapy. Examination of the specimen obtained in the first surgery revealed maxillary angiosarcoma, found to be positive for CD31, while negative for CD34, D2-40, and factor Ⅷ. Although no pathological residual tumor was observed after the planned wide surgery, cervical lymph node and distant metastases occurred. The patient died 24 months after the first surgery. Staining revealed VEGF receptor (VEGFR) 1, VEGFR2, phosphorylated Ak strain transforming, mitogen-activated protein kinase, and signal transducer and activator of transcription 3 positivity. Although our findings do not indicate that anti-VEGF therapy is beneficial for treating maxillary angiosarcomas, we found that VEGFR signaling pathways were activated in maxillary angiosarcomas similar to angiosarcomas originating at other sites. Herein, we report a case of maxillary angiosarcoma, focused on VEGFR and signaling pathway activation. To our knowledge, this is the first report to describe VEGFR system immunostaining findings in maxillary angiosarcoma.

Vascular endothelial growth factor receptor 2 contributes to shear stress-induced endothelial nitric oxide synthase expression in human lung microvascular endothelial cells

Introduction: Physiological shear stress (pSS) helps to maintain endothelial homeostasis via numerous intracellular signaling pathways. We previously showed that pSS upregulates mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in primary human lung microvascular endothelial cells (hLMVECs), and that phosphorylation of Akt (p-Akt), a phosphatidylinositol 3-kinase (PI3K) target, is increased. Upstream regulatory mechanisms for these responses have not been fully studied. Since vascular endothelial growth factor receptor 2 (VEGFR2) is an integral component of the mechanosensory complex that transduces shear stress-mediated signaling pathways in endothelial cells, we hypothesized that VEGFR2 is required for shear stress-induced eNOS expression and PI3K activation in hLMVECs. Methods: We cultured early passage (p≤7) primary hLMVECs from 4 unique donor lots in standard 6-well culture plates using a fixed volume (depth) and viscosity of tissue culture media. We exposed cells to shear stress for 24 hours using an orbital shaker and calculated the magnitude of shear stress exerted on hLMVECs using the extended solution to Stokes’ second problem. We defined the magnitude of pSS as 12 dyn/cm2. In some experiments, we exposed cells to pSS in the presence of chemical inhibitors of VEGFR2 (SU5416; 5 or 10 μM) or PI3K (LY294002; 10 μM). We collected whole cell lysates and performed immunoblotting to probe for phosphorylated eNOS (p-eNOS; Serine 1177), total eNOS, p-Akt (Serine 473), total Akt and housekeeping protein β-tubulin. Potential interactions between chemical inhibitors and pSS on protein expression and phosphorylation were analyzed by two-way ANOVA. Results: After 24 hours of pSS exposure, total eNOS levels were significantly (p<0.05) increased in hLMVECs, although the p-eNOS/eNOS ratio was not changed (p=0.85). Conversely, 24 hours of pSS exposure increased the p-Akt/Akt ratio (p<0.05) but did not increase total Akt levels (p=0.88). Inhibition of VEGFR2 activity with SU5416 attenuated pSS-induced changes in total eNOS levels (p<0.05 for interaction) but did not attenuate pSS-induced increases in the p-Akt/Akt ratio (p=0.91 for interaction). Inhibition of PI3K activity using LY294002 did not modify the effects of pSS on total eNOS levels (p=0.25 for interaction) or p-Akt/Akt ratio (p=0.47 for interaction). Conclusions: We conclude that VEGFR2 activity is necessary for pSS-mediated increases in eNOS protein levels, but not PI3K activity, in hLMVECs. Furthermore, neither VEGFR2 nor PI3K activity appear to be required for pSS-mediated eNOS phosphorylation in these cells. Collectively, these data suggest that shear stress regulates hLMVEC eNOS signaling via multiple signaling pathways. This complex regulation may have important implications for local nitric oxide bioavailability under pathological conditions that modify shear stress (e.g., pulmonary embolism, pulmonary hypertension). This work is supported by NIH grants HL126514 and HL159906. 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.

Targeting EGFR and VEGFR-2 Kinases With Nanoparticles: A Computational Approach for Cancer Therapy Advancement

The study investigates titanium and zinc nanoparticles as inhibitors for the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), pivotal regulators of cell processes. VEGFR-2 activation fuels tumor angiogenesis in cancer cells, sustaining malignant tissue expansion. Molecular docking analysis illustrates the nanoparticles’ binding to the active sites, inhibiting the phosphorylation of key proteins in downstream signaling. This inhibition offers a promising therapeutic approach to impede cancer-related signaling, potentially slowing down aberrant protein cascades controlled by EGFR and VEGFR-2. The findings propose a novel avenue for cancer treatment, targeting abnormal growth pathways using titanium and zinc nanoparticles.

β-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

β-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.

Data-driven derivation of an adverse outcome pathway linking vascular endothelial growth factor receptor (VEGFR), endocrine disruption, and atherosclerosis.

Dysregulation of Vascular Endothelial Growth Factor (VEGF) and its receptor (VEGFR) contributes to atherosclerosis and cardiovascular disease (CVD), making it a potential target for CVD risk assessment. High throughput screening (HTS) approaches have resulted in large-scale in vitro data, providing mechanistic information that can help assess chemical toxicity and identify molecular-initiating events (MIEs) of adverse outcome pathways (AOPs). AOPs represent a logical sequence of biological responses contributing to toxicity and are valuable tools to inform chemical risk assessments. Here, we used HTS data to formulate an AOP relating VEGF signaling perturbation to atherosclerosis. ToxCast, Tox21, and PubChem data were evaluated to obtain bio-profiles of 4165 compounds active in assays targeting VEGFR. Cheminformatics analysis identified 109 enriched structural fingerprints. Applying a subspace clustering approach based on chemical structure bioactivity yielded 12 primary targets, whose relevance to CVD was confirmed by an AI-assisted literature review. An AOP was hypothesized by coupling mechanistic relationships highlighted by HTS data with literature review findings, linking Serotonin Receptor (HTR), Estrogen Receptor Alpha (ERα), and Vasopressin Receptor (AVPR) targets with VEGFR activity, angiogenic signaling, and atherosclerosis. Several endocrine disrupting chemicals (EDCs), e.g., bisphenols, triclosan, dichlorodiphenyltrichloroethane (DDT), and polychlorinated biphenyls (PCBs), were identified as relevant chemical stressors. Subspace clustering of these chemicals evaluated potential MIEs and highlighted associations with use-case classes. By applying computational methods to profile HTS data and hypothesize a mechanistic AOP, this study proposes a data-driven approach to evaluating environmental cardiotoxicity, which could eventually supplement and reduce the need for animal testing in toxicological assessments.

Blood flow patterns switch VEGFR2 activity through differential S-nitrosylation and S-oxidation

Vascular endothelial growth factor receptor-2 (VEGFR2) plays a key role in maintaining vascular endothelial homeostasis. Here, we show that blood flows determine activation and inactivation of VEGFR2 through selective cysteine modifications. VEGFR2 activation is regulated by reversible oxidation at Cys1206 residue. H2O2-mediated VEGFR2 oxidation is induced by oscillatory flow in vascular endothelial cells through the induction of NADPH oxidase-4 expression. In contrast, laminar flow induces the expression of endothelial nitric oxide synthase and results in the S-nitrosylation of VEGFR2 at Cys1206, which counteracts the oxidative inactivation. The shear stress model study reveals that disturbed blood flow operated by partial ligation in the carotid arteries induces endothelial damage and intimal hyperplasia in control mice but not in knock-in mice harboring the oxidation-resistant mutant (C1206S) of VEGFR2. Thus, our findings reveal that flow-dependent redox regulation of the VEGFR2 kinase is critical for the structural and functional integrity of the arterial endothelium.

Antiangiogenic Action of JZL184 on Endothelial Cells via Inhibition of VEGF Expression in Hypoxic Lung Cancer Cells.

JZL184, an inhibitor of monoacylglycerol lipase (MAGL) and thus of the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), mediates various anticancer effects in preclinical studies. However, studies on the effect of this or other MAGL inhibitors under hypoxia, an important factor in tumor biology and response to cancer therapy, have not yet been performed in cancer cells. In the present study, the impact of the conditioned media (CM) of A549 and H358 lung cancer cells incubated with JZL184 under hypoxic conditions on the angiogenic properties of human umbilical vein endothelial cells (HUVECs) was investigated. Treatment of HUVECs with CM derived from cancer cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation compared with unconditioned medium, which was inhibited when cancer cells were incubated with JZL184. In this process, JZL184 led to a significant increase in 2-AG levels in both cell lines. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. Antiangiogenic and VEGF-lowering effects were also demonstrated for the MAGL inhibitor MJN110. Receptor antagonist experiments suggest partial involvement of the cannabinoid receptors CB1 and CB2 in the antiangiogenic and VEGF-lowering effects induced by JZL184. The functional importance of VEGF for angiogenesis in the selected system is supported by observations showing inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs by CM from hypoxic cancer cells treated with JZL184 or when hypoxic cancer cell-derived CM was spiked with a neutralizing VEGF antibody. On the other hand, JZL184 did not exert a direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. In conclusion, these results reveal a novel mechanism of antiangiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.

The co-expression pattern of VEGFR-2 with indicators related to proliferation, apoptosis, and differentiation of anagen hair follicles.

An increasing number of studies show that vascular endothelial growth factor is an important regulator of hair growth, and involves in processes of hair follicle development by vascularization. Recently, VEGF receptor-2 (VEGFR-2) has been detected in epithelial cells of hair follicles, indicating that it may have a direct role in the biological activity of hair follicles. To explore how VEGFR-2 regulates hair follicle development, we investigated the co-expression pattern of VEGFR-2 with β-catenin, Bax, Bcl-2, involucrin, AE13 (hair cortex cytokeratin), keratin 16, keratin 14, and Laminin 5 by immunofluorescence double staining in anagen hair follicles of normal human scalp skin. The results of double staining immunofluorescence showed a strong overlapping and similar expression pattern for VEGFR-2 with β-catenin and Bcl-2, and revealing associated expression pattern with involucrin, AE13, keratin 14, keratin 16, and Laminin 5. These results elucidated that VEGFR-2 activation may participate in hair follicle differentiation, proliferation, and apoptosis in vivo.

Deletion of Endothelial TRPV4 Protects Heart From Pressure Overload-Induced Hypertrophy.

Left ventricular hypertrophy is a bipolar response, starting as an adaptive response to the hemodynamic challenge, but over time develops maladaptive pathology partly due to microvascular rarefaction and impaired coronary angiogenesis. Despite the profound influence on cardiac function, the mechanotransduction mechanisms that regulate coronary angiogenesis, leading to heart failure, are not well known. We subjected endothelial-specific knockout mice of mechanically activated ion channel, TRPV4 (transient receptor potential cation channel subfamily V member 4; TRPV4ECKO) to pressure overload via transverse aortic constriction and examined cardiac function, cardiomyocyte hypertrophy, cardiac fibrosis, and apoptosis. Further, we measured microvascular density and underlying TRPV4 mechanotransduction mechanisms using human microvascular endothelial cells, ECM gels of varying stiffness, unbiased RNA sequencing, siRNA, Western blot, qPCR, and confocal immunofluorescence techniques. We demonstrate that endothelial-specific deletion of TRPV4 preserved cardiac function, cardiomyocyte structure, and reduced cardiac fibrosis compared with TRPV4lox/lox mice, 28 days post-transverse aortic constriction. Interestingly, comprehensive RNA sequencing analysis revealed an upregulation of proangiogenic factors (VEGFα NOS3, and FGF2) with concomitant increase in microvascular density in TRPV4ECKO hearts after transverse aortic constriction compared with TRPV4lox/lox. Further, an increased expression of VEGFR2 (vascular endothelial growth factor receptor 2) and activation of the YAP (yes-associated protein) pathway were observed in TRPV4ECKO hearts. Mechanistically, we found that downregulation of TRPV4 in endothelial cells induced matrix stiffness-dependent activation of YAP and VEGFR2 via the Rho/Rho kinase/LATS pathway. Our results suggest that endothelial TRPV4 acts as a mechanical break for coronary angiogenesis, and uncoupling endothelial TRPV4 mechanotransduction attenuates pathological cardiac hypertrophy by enhancing coronary angiogenesis.

Novel azole-urea hybrids as VEGFR-2 inhibitors: Synthesis, in vitro antiproliferative evaluation and in silico studies

The vascular endothelial growth factor receptor-2 (VEGFR-2) is a receptor tyrosine kinase known to be abnormally expressed in various malignant tumors, including breast cancer, and is considered one of the most important contributors to tumor angiogenesis. Sorafenib is one of many VEGFR-2 inhibitors that have received approval for clinical use from the US FDA in recent years. Accordingly, in this study, the synthesis of two new pyrazoles, six 1,3,4-oxadiazoles, four 1,3,4-thiadiazoles, and ten 1,2,4-triazole-3-thione derivatives having structural characteristics similar to sorafenib was carried out. A preliminary screening of synthesized compounds and known inhibitors sorafenib and staurosporine at 10 µM concentration on in vitro activity of VEGFR-2 was performed, and compounds 10c, 8a, and 11 g were identified as the most potent derivatives with% VEGFR-2 residual activities lower than 30%, and dose-dependent inhibition studies was carried out to determine the IC50 values of these inhibitors. Compound 10c was found to be the most potent inhibitor of VEGFR-2 activity with an IC50 value of 0.664 µM. The anti-proliferative activity of synthesized derivatives was assessed against a breast carcinoma (MCF-7) cell line, a triple negative human breast adenocarcinoma (MDA-MB-231) cell line, and noncancerous fibroblast cells (L929). Compound 8a displayed superior activity when compared to sorafenib against MCF-7 (7.69 fold) and MDA-MB-231 (1.52 fold) cell lines while displaying 3.75-fold less toxicity against the normal L929 cell line. Annexin V binding assay revealed that compound 8a significantly increased early and late apoptosis in MCF-7 cells and late apoptosis and necrosis in MDA-MB-231 cells. Computational studies such as molecular docking and ADMET evaluation were performed to elucidate the binding interactions and drug-likeness of the synthesized compounds. The results indicate that compound 8a could be a promising candidate for the development of a novel anti-angiogenic and anti-proliferative agent.

Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes

Low circulating phosphate (Pi) leads to rickets, characterized by expansion of the hypertrophic chondrocytes (HCs) in the growth plate due to impaired HC apoptosis. Studies in HCs demonstrate that Pi activates the Raf/MEK/ERK1/2 and mitochondrial apoptotic pathways. To determine how Pi activates these pathways, a small-molecule screen was undertaken to identify inhibitors of Pi-induced ERK1/2 phosphorylation in HCs. Vascular endothelial growth factor receptor 2 (VEGFR2) was identified as a target. Invitro studies in HCs demonstrate that VEGFR2 inhibitors block Pi-induced pERK1/2 and caspase-9 cleavage. Like Pi, rhVEGF activates ERK1/2 and caspase-9 in HCs and induces phosphorylation of VEGFR2, confirming that Pi activates this signaling pathway in HCs. Chondrocyte-specific depletion of VEGFR2 leads to an increase in HCs, impaired vascular invasion, and a decrease in HC apoptosis. Thus, these studies define a role for VEGFR2 in transducing Pi signals and mediating its effects on growth plate maturation.

BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

Novel imino-thiazoloquinoxaline derivatives against renal cell carcinoma: less radiation-damaging approach

Renal cell carcinoma (RCC) is the most fatal tumor in the urinary system. Resistance development and unmet effective responses, request new anticancer agents with better therapeutic index. Ten new imino-thiazolo-quinoxaline derivatives (5a-j) were synthesized and preliminary evaluated for downregulation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) activity taking sorafenib as a reference drug. Compounds 5d & 5h showed potent inhibition to VEGFR-2 activity at IC50 89.35 nM & 60.64 nM, respectively, then they both were further evaluated in-vitro against urinary bladder cancer cell line T-24 taking sorafenib as a reference drug. Compound 5h displayed nearly anticancer activity to sorafenib against T-24 cell line in all concentrations tested except at concentration 10 µM where it highly suppressed cell viability to 6.71 % compared to 15.15% of sorafenib. Compound 5h was then evaluated for its ameliorative efect against radiation induced renal tissue injury. Assessment of pro-angiogenic (VEGFR-2), pro-fibrotic (transforming growth factor-beta 1 (TGF-β1)) and apoptotic (caspase-3) markers, as well as histopathological examinations were performed on kidney of irradiated mice. Results showed ability of compound 5h to downregulate VEGFR-2 activity and its cytotoxic effect against RCC, in addition to mitigation of radiation induced renal tissue injury. Ethyl imino-thiazoloquinoxaline carboxylate derivative 5h showed a potential cytotoxic activity against RCC and could be considered a promosing alleviative candidate when employed post radiotherapy regimen.Graphical

Elucidating activation and deactivation dynamics of VEGFR-2 transmembrane domain with coarse-grained molecular dynamics simulations.

The vascular endothelial growth factor receptor 2 (VEGFR-2) is a member of receptor tyrosine kinases (RTKs) and is a dimeric membrane protein that functions as a primary regulator of angiogenesis. As is usual with RTKs, spatial alignment of its transmembrane domain (TMD) is essential toward VEGFR-2 activation. Experimentally, the helix rotations within TMD around their own helical axes are known to participate importantly toward the activation process in VEGFR-2, but the detailed dynamics of the interconversion between the active and inactive TMD forms have not been clearly elucidated at the molecular level. Here, we attempt to elucidate the process by using coarse grained (CG) molecular dynamics (MD) simulations. We observe that inactive dimeric TMD in separation is structurally stable over tens of microseconds, suggesting that TMD itself is passive and does not allow spontaneous signaling of VEGFR-2. By starting from the active conformation, we reveal the mechanism of TMD inactivation through analyzing the CG MD trajectories. We observe that interconversions between a left-handed overlay and a right-handed one are essential for the process of going from an active TMD structure to the inactive form. In addition, our simulations find that the helices can rotate properly when the overlaying structure of the helices interconverts and when the crossing angle of the two helices changes by larger than ~40 degrees. As the activation right after the ligand attachment on VEGFR-2 will take place in the reverse manner of this inactivation process, these structural aspects will also appear importantly for the activation process. The rather large change in helix configuration for activation also explains why VEGFR-2 rarely self-activate and how the activating ligand structurally drive the whole VEGFR-2. This mechanism of TMD activation / inactivation within VEGFR-2 may help in further understanding the overall activation processes of other RTKs.

SENP6-Mediated deSUMOylation of VEGFR2 Enhances Its Cell Membrane Transport in Angiogenesis.

Angiogenesis is a significant pathogenic characteristic of diabetic microangiopathy. Advanced glycation end products (AGEs) are considerably elevated in diabetic tissues and can affect vascular endothelial cell shape and function. Regulation of the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway is a critical mechanism in the regulation of angiogenesis, and VEGFR2 activity can be modified by post-translational changes. However, little research has been conducted on the control of small ubiquitin-related modifier (SUMO)-mediated VEGFR2 alterations. The current study investigated this using human umbilical vein endothelial cells (HUVECs) in conjunction with immunoblotting and immunofluorescence. AGEs increased Nrf2 translocation to the nucleus and promoted VEGFR2 expression. They also increased the expression of sentrin/SUMO-specific protease 6 (SENP6), which de-SUMOylated VEGFR2, and immunofluorescence indicated a reduction in VEGFR2 accumulation in the Golgi and increased VEGFR2 transport from the Golgi to the cell membrane surface via the coatomer protein complex subunit beta 2. VEGFR2 on the cell membrane was linked to VEGF generated by pericytes, triggering the VEGF signaling cascade. In conclusion, this study demonstrates that SENP6 regulates VEGFR2 trafficking from the Golgi to the endothelial cell surface. The SENP6-VEGFR2 pathway plays a critical role in pathological angiogenesis.

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5β1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

Thioredoxin-Interacting Protein Inhibited Vascular Endothelial Cell-Induced HREC Angiogenesis Treatment of Diabetic Retinopathy.

Diabetic retinopathy is the most common reason for blindness among employed adults worldwide. Hyperglycemia and the overaccumulation of vascular endothelial growth factor (VEGF) lead to diabetic retinopathy, pathological angiogenesis in diabetic retinopathy, and consequent visual impairment. Expression levels of thioredoxin-interacting protein (TXNIP) substantially increase in retinal endothelial cells in diabetic circumstances. The part of TXNIP in retinal angiogenesis combined with diabetes remains unclear. This study examined the effect of reduced TXNIP expression levels and determined how it affects diabetic retinal angiogenesis. Display of human retinal vascular endothelial cells (HRECs) to moderately high glucose (MHG) encouraged tube formation and cell migration, not cell proliferation. In response to MHG conditions, in HRECs, TXNIP knockdown inhibited the production of reactive oxygen species (ROS), cell migration, tube formation, and the Akt/mTOR activation pathway. In addition, gene silencing of TXNIP decreased the VEGF-triggered angiogenic response in HRECs by preventing activation of both VEGF receptor 2 and the downstream components of the Akt/mTOR pathway signaling. Furthermore, TXNIP knockout mice reduced VEGF-induced or VEGF- and MHG-triggered ex vivo retinal angiogenesis compared to wild-type mice. Finally, our findings revealed that TXNIP knockdown suppressed VEGF- and MHG-triggered angiogenic responses in HRECs and mouse retinas, indicating that TXNIP is a promising therapeutic window against the proliferation of diabetic patients' retinopathy.

Endothelial-Specific Expression of CIDEC Improves High-Fat Diet-Induced Vascular and Metabolic Dysfunction.

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid droplet-associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells and regulates vascular function. The current study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against high-fat diet-induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, these mice displayed improved insulin signaling and endothelial nitric oxide synthase activation, enhanced endothelium-dependent vascular relaxation, and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-vascular endothelial growth factor A (VEGFA)-vascular endothelial growth factor receptor 2 (VEGFR2) that reduced VEGFA and VEGFR2 degradation, thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggest that CIDEC may be investigated as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.

Angiostatic freeze or angiogenic move? Acute cold stress prevents angiokine secretion from murine myotubes but primes primary endothelial cells for greater migratory capacity.

The skeletal muscle tissue can adapt to exercise and environmental stressors with a remarkable plasticity. Prolonged cold stress exposure has been associated to increased skeletal muscle capillarization. Angioadaptation refers to the coordinated molecular and cellular processes that influence the remodeling of skeletal muscle microvasculature. Two cell types are central to angioadaptation: the myocytes, representing an important source of angiokines; and the skeletal muscle endothelial cell (SMECs), targets of these angiokines and main constituents of muscle capillaries. The influence of cold stress on skeletal muscle angioadaptation remains largely unknown, particularly with respect to myocyte-specific angiokines secretion or endothelial cell angioadaptive responses. Here, we use an in vitro model to investigate the impact of cold stress (28°C versus 37°C) on C2C12 myotubes and SMECs. Our main objectives were to evaluate: 1) the direct impact of cold stress on C2C12 cellular expression of angiokines and their release in the extracellular environment; 2) the indirect impact of cold stress on SMECs migration via these C2C12-derived angiokines; and 3) the direct effect of cold stress on SMECs angioadaptive responses, including migration, proliferation, and the activation of the vascular endothelial growth factor receptor-2 (VEGFR2). Cold stress reduced the secretion of angiokines in C2C12 myotubes culture media irrespective their pro-angiogenic or angiostatic nature. In SMECs, cold stress abrogated cell proliferation and reduced the activation of VEGFR2 despite a greater expression of this receptor. Finally, SMECs pre-conditioned to cold stress displayed an enhanced migratory response when migration was stimulated in rewarming conditions. Altogether our results suggest that cold stress may be overall angiostatic. However, cold stress accompanied by rewarming may be seen as a pro-angiogenic stressor for SMECs. This observation questions the potential for using pre-cooling in sport-performance or therapeutic exercise prescription to enhance skeletal muscle angioadaptive responses to exercise.