Human Vascular Endothelial Growth Factor Research Articles

Development of a system of heparin multilayers on titanium surfaces for dual growth factor release.

The aim of the present study was to establish a modular platform of poly-L-lysine-heparin (PLL-Hep) polyelectrolyte multilayer (PEM) coatings on titanium surfaces for dual growth factor delivery of recombinant human bone morphogenic protein 2 (rhBMP2) and recombinant human vascular endothelial growth factor 165 (rhVEGF165) in clinically relevant quantities. Release characteristics for both growth factors differed significantly depending on film architecture. rhBMP2 induced activation of alkaline phosphatase in C2C12 cells and proliferation of human mesenchymal stem cells (hMSCs). rhVEGF mediated induction of von Willebrand factor (vWF) in hMSCs and proliferation of human umbilical vein endothelial cells. Osteogenic and angiogenic effects were modified by variation in cross-linking and architecture of the PEMs. By creating multilayer films with distinct zones, release characteristics and proportion of both growth factor delivery could be tuned and surface-activity modified to enhance angiogenic or osteogenic function in various ways. In summary, the system provides a modular platform for growth factor delivery that allows for individual composition and accentuation of angiogenic and osteogenic surface properties.

Phase II Study of Ramucirumab Plus Irinotecan Combination Therapy as Second-Line Treatment in Patients with Advanced Gastric Cancer: HGCSG1603

BackgroundRamucirumab is a human IgG1 monoclonal vascular endothelial growth factor receptor-2 antibody that inhibits tumor cell growth and affects the tumor cell microenvironment. We assessed the efficacy and safety of ramucirumab plus irinotecan combination therapy as second-line treatment in patients with previously treated advanced gastric cancer.Materials and MethodsPatients with advanced gastric cancer refractory or intolerant to primary chemotherapy were included. Ramucirumab 8 mg/kg plus irinotecan 150 mg/m2 combination therapy was administered every 2 weeks. The primary endpoint was progression-free survival rate at 6 months and secondary endpoints were overall survival, progression-free survival, response rate, safety, and dose intensity for each drug.ResultsThirty-five patients were enrolled between January 2018 and September 2019. The progression-free survival rate at 6 months was 26.5% [95%CI, 13.2%–41.8%, P = .1353)]. Median progression-free and overall survivals were 4.2 months (95%CI, 2.5-5.4 months) and 9.6 months (95%CI, 6.4-16.6 months), respectively. The overall response rate was 25.9% (95%CI, 11.1-36.3%) and disease control rate was 85.2% (95%CI, 66.3-95.8%). Grade ≥3 adverse events that occurred in >10% of patients included neutropenia, leucopenia, anemia, anorexia, and febrile neutropenia. No death or new safety signals with a causal relation to the study treatment were observed.ConclusionAlthough the primary endpoint was not achieved statistically, combination therapy of ramucirumab plus irinotecan showed anticancer activity and a manageable safety profile for second-line treatment of patients with advanced gastric cancer.

Relationship Between Endothelial and Angiogenesis Biomarkers Envisage Mortality in a Prospective Cohort of COVID-19 Patients Requiring Respiratory Support.

PurposeEndothelial damage and angiogenesis are fundamental elements of neovascularisation and fibrosis observed in patients with coronavirus disease 2019 (COVID-19). Here, we aimed to evaluate whether early endothelial and angiogenic biomarkers detection predicts mortality and major cardiovascular events in patients with COVID-19 requiring respiratory support.MethodsChanges in serum syndecan-1, thrombomodulin, and angiogenic factor concentrations were analysed during the first 24 h and 10 days after COVID-19 hospitalisation in patients with high-flow nasal oxygen or mechanical ventilation. Also, we performed an exploratory evaluation of the endothelial migration process induced by COVID-19 in the patients' serum using an endothelial cell culture model.ResultsIn 43 patients, mean syndecan-1 concentration was 40.96 ± 106.9 ng/mL with a 33.9% increase (49.96 ± 58.1 ng/mL) at day 10. Both increases were significant compared to healthy controls (Kruskal–Wallis p < 0.0001). We observed an increase in thrombomodulin, Angiopoietin-2, human vascular endothelial growth factor (VEGF), and human hepatocyte growth factor (HGF) concentrations during the first 24 h, with a decrease in human tissue inhibitor of metalloproteinases-2 (TIMP-2) that remained after 10 days. An increase in human Interleukin-8 (IL-8) on the 10th day accompanied by high HGF was also noted. The incidence of myocardial injury and pulmonary thromboembolism was 55.8 and 20%, respectively. The incidence of in-hospital deaths was 16.3%. Biomarkers showed differences in severity of COVID-19. Syndecan-1, human platelet-derived growth factor (PDGF), VEGF, and Ang-2 predicted mortality. A multiple logistic regression model with TIMP-2 and PDGF had positive and negative predictive powers of 80.9 and 70%, respectively, for mortality. None of the biomarkers predicted myocardial injury or pulmonary thromboembolism. A proteome profiler array found changes in concentration in a large number of biomarkers of angiogenesis and chemoattractants. Finally, the serum samples from COVID-19 patients increased cell migration compared to that from healthy individuals.ConclusionWe observed that early endothelial and angiogenic biomarkers predicted mortality in patients with COVID-19. Chemoattractants from patients with COVID-19 increase the migration of endothelial cells. Trials are needed for confirmation, as this poses a therapeutic target for SARS-CoV-2.

Structural Characterization of RC28-E, a Recombinant Fusion Protein With Dual Targets on VEGF and FGF2

Vascular endothelial growth factor (VEGF) and fibroblast growthfactor (FGF) play important roles in angiogenesis-related diseases. RC28-E is a soluble fusion protein composed of the human VEGF receptor 1 (VEGFR1) extracellular domain 2 (ECD 2), VEGFR2 ECD 3, FGFR1 ECDs 2 and 3, and the Fc regions of human immunoglobulin G1. By targeting both VEGF and FGF2, RC28-E may represent a useful antiangiogenetic agent, but structural and functional characterizations of this fusion protein are needed. Liquid chromatography–tandem mass spectrometry, size exclusion high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing, and bio-layer interferometry were used to characterize the properties of RC28-E. The purity of RC28-E was confirmed to be 98% or greater. The glycosylation modification of RC28-E was found to be very complicated, with 11 potential N-linked glycosylation points and 23 types of N-glycans, causing high heterogeneity of the protein. The primary modifications of the amino acid sequence of RC28-E protein included C-terminal K truncation, N-deamidation, and M-oxidation modification. Notably, RC28-E demonstrated a higher affinity for both VEGF and FGF2 than VEGF trap or FGF trap for their respective targets.

Potential mechanisms involved on how systemic IgG antibodies specific to vascular endothelial growth factor (VEGF) and induced by active immunotherapy decrease platelet derived free-VEGF

CIGB-247 is a vascular endothelial growth factor (VEGF)-based active immunotherapy and it is currently under investigation for cancer treatment. This specific active immunotherapy encompasses two vaccine candidates that use a human VEGF variant molecule as antigen, in combination with two clinically tested adjuvants: VSSP or aluminum phosphate. CIGB-247 has been evaluated in patients with advanced solid tumors, recruited in two phase I clinical trials, and it has been shown to be safe and immunogenic by activating both cellular and humoral immune responses against human VEGF. The immunization induces specific IgG antibodies, and also shows as effect, the reduction of free-VEGF levels within platelets (platelet-derived free VEGF). The production of systemic IgG antibodies and the presence of VEGF in another compartment, almost exclusively within platelets, have arisen some questions about this effect detected in the vaccinated-cancer patients. Based on some relevant published works about platelet endocytosis and VEGF pharmacodynamics during bevacizumab treatment as well as the phase I clinical data of CIGB-247, this investigation aims to hypothesize and analyze the potential mechanisms involved in the reduction of platelet-derived free VEGF as a result of vaccination with CIGB-247. Abbreviations: FcγR: Fc gamma receptors; IC: immune complexes; VEGF: vascular endothelial growth factor; VEGFR1: vascular endothelial growth factor receptor 1; VEGFR2: vascular endothelial growth factor receptor 2.

Beneficial Effects of Caffeic Acid Phenethyl Ester on Wound Healing in a Diabetic Mouse: Role of VEGF and NO

Cutaneous wound healing is delayed in patients with diabetes. Caffeic acid phenethyl ester (CAPE) has been identified as an effective constituent of propolis with improved wound healing abilities via an oxidative stress decrease. However, its impact on wound healing in diabetic models and its underlying mechanisms remain unclear. Determining the vascular endothelial growth factor (VEGF) contents in a human vascular smooth muscle cell (VSMC)-conditioned medium was assessed using human VEGF immunoassay and vascular reactivity using porcine coronary artery rings. Later, C57BL/6 or db/db mice were anesthetized, after which a 6-mm biopsy punch was manipulated for perforation via the back skin. Subsequently, CAPE was applied to the wound and changed daily. Furthermore, the injury in each mouse was digitally photographed, and the wound area was quantified. We observed that CAPE increased VEGF levels in human VSMC-conditioned medium, improved endothelium-dependent nitric oxide (NO)-mediated vasorelaxation, inhibited U46619-induced vasoconstriction porcine coronary artery, and enhanced cutaneous wound healing in the diabetic mouse model. Hence, we propose that CAPE improves wound healing in diabetic mice, which is aided by increased VEGF and NO expression.

Vascular cell behavior on glycocalyx–mimetic surfaces: Simultaneous mimicking of the chemical composition and topographical structure of the vascular endothelial glycocalyx

The endothelial glycocalyx is a carbohydrate–rich layer overlying the outermost surface of endothelial cells. It mediates intercellular interactions by specific chemical compositions (e.g., proteoglycans containing glycosaminoglycan (GAG) side chains) and micro/nanotopography. Inspired by the endothelial glycocalyx, we fabricated a series of glycocalyx–mimetic surfaces with tunable chemical compositions (GAG–like polymers with different functional units) and topographical structures (micro/nanopatterns with pillars different in size). The combination of micro/nanopatterns and GAG–like polymers was flexibly and precisely controlled by replica molding using silicon templates (Si templates) and visible light–initiated polymerization. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs) were suppressed on surfaces modified with polymers of 2–methacrylamido glucopyranose (MAG) but promoted on surfaces modified with polymers of sodium 4–vinyl–benzenesulfonate (SS) and copolymers of SS and MAG. Surface micro/nanopatterns showed highly complicated effects on surfaces grafted with different GAG–like polymers. Moreover, the spread of HUVSMCs was highly promoted on all flat/patterned surfaces containing sulfonate units, and the elongation effect was stronger on surfaces with smaller pillars. On all the flat/patterned surfaces modified with GAG–like polymers, the adsorption of human vascular endothelial growth factor (VEGF) and human basic fibroblast growth factor (bFGF) was improved, and the amount of VEGF and bFGF absorbed on patterned surfaces containing sulfonate units decreased with pattern dimensions. The decreasing trend of VEGF and bFGF adsorption was in accordance with HUVEC density, suggesting that glycocalyx–mimetic surfaces influence the adsorption of VEGF and bFGF and further influence the growth behavior of vascular cells.

Role of Micro-RNA in the Regulation of Cell Polarization in Hepatocellular Carcinoma

The occurrence of tissue scarring and architecture-modifying signaling led to a tumorigenic microenvironment. Targeting specifically the biological mediators responsible for the physiological and morphological changes accommodating hepatocellular carcinoma (HCC) growth may be the key for identifying a future HCC cure. Morphological and physiological features of cultured HepG2 cells in both stimulated recombinant human vascular endothelial growth factor (VEGF165) and unstimulated (control) conditions were assessed. Quantitative reverse transcription (RT)-PCR measured endogenous VEGF expression levels. The assessment of proangiogenic biological mediator (micro RNA [miR]-296, miR-31, and miR-17) profiles was achieved by polarization-inducing VEGF165 stimulation followed by quantitative RT-PCR. In vitro conditions reproduced successfully the physiological environment leading to the occurrence of HCC, including the successful HepG2 polarization following VEGF stimulation. While endogenous VEGF production only occurs if complete polarization has been reached, the quantified biological mediator profiles determined here pointed at either possible early stages of depolarization or at the lack of tumorigenic potential of the HepG2 cells. All tested miRs displayed upregulated profiles, although miR-296 was less amplified (3.78-fold compared with control) than miR-31 or miR-17 (6.5- and 6.6-fold, respectively). The findings surrounding miR-17 reproduce similar data reported in the literature; the unexpected high miR-31 expression was intriguing. Given HepG2 cells' minimal tumorigenic potential, the unexpected multifold upregulation of miR-31 may be a cause or a consequence of HepG2 cells' low tumorigenic potential. The exploration of miR-31 therapeutic potential may be a future rewarding endeavor.

Effects of root-end filling materials on vascular endothelial cell proliferation and tube formation

Background/purposeRegarding root-end filling materials in apical surgery, sealing ability and biocompatibility are useful for treatment. Angiogenesis, which occurs in the process of periapical wound healing, is closely related to bone formation. In this study, we investigated the effects of root-end filling materials on vascular endothelial cell proliferation and angiogenesis. Materials and methodsMineral trioxide aggregate (MTA), 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin, Super EBA, and CS-BG-multi, bioactive glass-related materials, were used. After curing, each material was soaked in a medium for 1 or 7 days, and then cultured for 1–7 days to investigate the effects on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis, and vascular endothelial growth factor receptors (VEGFRs) mRNA expression. ResultsIn the 1-day soaked sample, there was significantly less proliferation in MTA and Super EBA on day 7 of culture. In the 7-day soaked sample, there was significantly less proliferation in Super EBA and CS-BG-multi on day 7 of culture. Tube formation was significantly high in MTA in both the 1-day and 7-day soaked samples, significantly high in SB in the 1-day soaked sample, and significantly low in Super EBA in both the 1-day and 7-day soaked samples. CS-BG-multi was comparable to the control. VEGFR-1 and VEGFR-2 mRNA expressions showed an upward trend in MTA, and a trend similar to the control in SB. ConclusionMTA and 4-META/MMA-TBB resin had a higher pro-angiogenic effect while Super EBA had a less pro-angiogenic effect. CS-BG-multi had low toxicity on tube formation of HUVEC.

Elucidation of optimal proteinuria management based on the risk of ramucirumab-induced proteinuria.

Proteinuria is a dose-limiting adverse effect of ramucirumab treatment, which is an anti-angiogenic agent that targets the human vascular endothelial growth factor. The predictors of proteinuria have not been completely elucidated and there is currently no consensus. The present study aimed to identify the risk factors for ramucirumab-induced proteinuria and to determine an optimal proteinuria management. A total of 145 patients who received ramucirumab at Ogaki Municipal Hospital (Ogaki, Japan) between September 2015 and March 2021 were retrospectively studied. Multivariate logistic regression analysis was conducted to evaluate the association between the patient baseline characteristics and the development of proteinuria following ramucirumab treatment. Furthermore, the time of proteinuria onset and of the worst qualitative proteinuria were recorded. Proteinuria (>2+) following ramucirumab was independently associated with lung cancer [odd ratio (OR): 0.232, 95% confidence interval (CI): 0.061-0.874; P=0.031] and proteinuria at the start of treatment [qualitative test (+/-); OR: 4.760, 95% CI: 1.360-16.700; P=0.041]. The median time of onset of proteinuria was 56 days (time range, 7-414 days), and the median time when the worst qualitative results were observed was 83 days (time range, 7-442 days). The >2+ proteinuria in the qualitative test was observed in 27 out of the 82 patients with gastric cancer (P=0.041), 8/21 patients with colon cancer (P=0.188), and in 3 out of the 37 patients with lung cancer (P=0.003). The prevalence of proteinuria was low in patients with lung cancer, and proteinuria (>2+) was likely to occur when the proteinuria at the start of ramucirumab was (+/-) by qualitative test. The results from the present study indicated that particular attention should be paid to proteinuria at the start of treatment when monitoring proteinuria as an adverse event of ramucirumab treatment.

Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Abstract 11090: Therapeutic Effect of VEGF- and SDF-Nanoparticles in the Rat Monocrotaline Model of Pulmonary Hypertension

Introduction: Pulmonary vascular endothelial cell injury, dysfunction, and apoptosis are associated with the development of pulmonary arterial hypertension (PAH). Improving endothelial function may be therapeutically beneficial in this disorder. In the present study, we examined the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), proteins that support endothelial repair and tissue regeneration, in rats with monocrotaline-induced PAH. A polysaccharide nanoparticle was used as a carrier for VEGF and SDF-1α delivery, the formulation of which extends the retention time of the proteins in the lung by ~17-fold compared with free protein. Method: A crosslinked dextran sulfate-chitosan nanoparticle (XDSCS NP) was prepared as the carrier. Recombinant human VEGF and SDF-1α were incorporated in the XDSCS NP to generate VEGF-NP and SDF-NP, respectively. Athymic nude rats were injected with monocrotaline (50 mg/kg, IP) to induce PAH. Two weeks after the injection, VEGF-NP (8 μg/rat) and SDF-NP (4 μg/rat) were delivered to the lungs of rats via intratracheal aerosolization. At 4 weeks, catheterization of right and left heart was performed, and lung tissue collected for histological analysis. Results: Monocrotaline induced PAH in the nude rats, leading to right ventricular systolic pressure (RVSP) of 71±8 mmHg (mean±SD, n=8) vs. 29±2 mmHg in the untreated control rats. VEGF-NP and SDF-NP treatment reduced the RVSP to 48±13 mmHg. The Fulton indices of the three groups were 0.45±0.07, 0.22±0.01, and 0.25±0.06, respectively. The pulmonary vascular resistance indices of the groups were 3.2±0.7, 0.6±0.3, and 2.1±1.0 mmHg.min/ml/kg, respectively. Histological analysis showed monocrotaline-treated rats had prominent muscularization of precapillary vessels, which was markedly reduced by the VEGF-NP and SDF-NP treatment. Aerosolization of free VEGF and SDF-1α or of empty XDSCS NP carrier had no effect on monocrotaline-induce PAH. Conclusion: These data indicate that the delivery of VEGF and SDF to the lungs of rats in a NP-bound form had beneficial therapeutic effects in monocrotaline-induced PAH, and offer the potential for a novel therapeutic strategy for human pulmonary hypertensive disorders.

A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation.

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5′-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

Enhanced Neovascularization Using Injectable and rhVEGF‐Releasing Cryogel Microparticles

Front Cover: This article discusses developing a growth factor-releasing cryogel microparticle system and it's the therapeutic efficacy of human vascular endothelial growth factor for neovascularization. The cryogels are fabricated as micro-sized particles, and can thus be extruded through a needle to deliver. This is reported by Mihn Jeong Park, Young-Hyeon An, Young Hwan Choi, Hwan D. Kim and Nathaniel S. Hwang in article number 2100234.

Apatinib inhibits the growth of small cell lung cancer via a mechanism mediated by VEGF, PI3K/Akt and Ki-67/CD31.

This study aimed to investigate the anti‐tumour effect of apatinib on extensive‐stage small cell lung cancer (SCLC) and elucidate the associated mechanisms. NCI‐H345 cells were selected as model cells because of high expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and phosphorylated‐VEGFR2 (pVEGFR2). Cells were exposed to recombinant human VEGF (rhVEGF) and apatinib. Cells were then divided into eight groups, namely, control, rhVEGF, apatinib, rhVEGF+apatinib, serum‐free medium (SM), SM+rhVEGF, SM+apatinib and SM+rhVEGF+apatinib. In comparison with the control group, cell proliferation in vitro in apatinib, SM, SM+apatinib and SM+rhVEGF+apatinib groups was inhibited, particularly in SM+apatinib group. The effect of apatinib on tumour growth in vivo was investigated using a mouse xenograft tumour model. In comparison with the control group, tumour sizes were reduced in apatinib‐treated group on days 34 and 37. Immunohistochemical and immunofluorescence staining revealed that VEGF, pVEGFR2, PI3K, AKT, p‐ERK1/2, Ki‐67 and CD31 in the tumour cells of apatinib‐treated group were downregulated compared with control group. Haematoxylin and eosin staining revealed that apatinib promoted the necrosis of SCLC cells in vivo. In conclusion, apatinib inhibited the growth of SCLC cells by downregulating the expression of VEGF, pVEGFR2, p‐PI3K, p‐AKT, p‐ERK1/2, Ki‐67 and CD31.

AAV-Mediated Expression of Human VEGF, TNF-α, and IL-6 Induces Retinal Pathology in Mice.

PurposeRetinopathies display complex pathologies, including vasculopathies, inflammation, and fibrosis, leading ultimately to visual impairment. However, animal models accurately reflecting these pathologies are lacking. In this study, we evaluate the suitability of using Adeno-associated virus (AAV)-mediated long-term expression of cytokines to establish retinal pathology in the murine retina.MethodsWe administered recombinant, Müller-glia targeted AAV-ShH10 into the mouse vitreous to induce retinal expression of either human vascular endothelial growth factor (VEGF)-A165, tumor necrosis factor alpha (TNF-α), or interleukin-6 (IL-6) and evaluated consequent effects by optical coherence tomography, fluorescein angiography, and histology.ResultsIntravitreal injection of AAVs resulted in rapid and stable expression of the transgenes within 1 to 6 weeks. Akin to the role of VEGF-A in wet age-related macular degeneration, expression of VEGF-A led to several vasculopathies in mice, including neovascularization and vascular leakage. In contrast, the expression of the proinflammatory cytokines TNF-α or IL-6 induced retinal inflammation, as indicated by microglial activation. Furthermore, the expression of TNF-α, but not of IL-6, induced immune cell infiltration into the vitreous as well as vasculitis, and subsequently induced the development of fibrosis and epiretinal membranes.ConclusionsIn summary, the long-term expression of human VEGF-A165, TNF-α, or IL-6 in the mouse eye induced specific pathologies within 6 weeks that mimic different aspects of human retinopathies.Translational RelevanceAAV-mediated expression of human genes in mice is an attractive approach to provide valuable insights into the underlying molecular mechanisms causing retinopathies and is easily adaptable to other genes and preclinical species supporting drug discovery for retinal diseases.

Microneedle-mediated vascular endothelial growth factor delivery promotes angiogenesis and functional recovery after stroke

Ischemic stroke is still the major cause of disability worldwide. Although vascular endothelial growth factor (VEGF) is able to promote both angiogenesis and functional recovery, its use is limited by needle-induced injury, nonhomogenous VEGF distribution, and limited VEGF retention in the brain after intracranial or intravenous injection. Here, we first present a gelatin methacryloyl (GelMA) microneedle (MN)-based platform for the sustained and controlled local delivery of an adeno-associated virus (AAV) expressing human VEGF (AAV-VEGF) that achieves homogenous distribution and high transfection efficiency in ischemic brains. An ischemic stroke model was established in adult rats, and MNs loaded with AAV-VEGF were epicortically inserted into both the ischemic core and penumbra of these rats one day after the onset of ischemia. One week later, the inflammatory response and microneedle biocompatibility were assessed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Eight weeks later, angiogenesis and neural stem cell proliferation and migration were assessed. GelMA MN implantation did not elicit an obvious inflammatory response and had good biocompatibility in the brain. AAV–green fluorescent protein (GFP)-loaded MNs could achieve successful transfection and homogeneous distribution in the brain cortex three weeks postoperatively. MNs loaded with AAV-VEGF increased VEGF expression and enhanced functional angiogenesis and neurogenesis. In summary, MNs might emerge as a promising platform for delivering various therapeutics to treat ischemic stroke and repair other neurologically diseased tissues.

A novel conjunctive microenvironment derived from human subcutaneous adipose tissue contributes to physiology of its superficial layer

BackgroundIn human subcutaneous adipose tissue, the superficial fascia distinguishes superficial and deep microenvironments showing extensions called retinacula cutis. The superficial subcutaneous adipose tissue has been described as hyperplastic and the deep subcutaneous adipose tissue as inflammatory. However, few studies have described stromal-vascular fraction (SVF) content and adipose-derived stromal/stem cells (ASCs) behavior derived from superficial and deep subcutaneous adipose tissue. In this study, we analyzed a third conjunctive microenvironment: the retinacula cutis superficialis derived from superficial subcutaneous adipose tissue.MethodsThe samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery in France (Declaration DC-2008-162) and Brazil (Protocol 145/09).ResultsThe SVF content was characterized in situ by immunofluorescence and ex vivo by flow cytometry revealing a high content of pre-adipocytes rather in superficial subcutaneous adipose tissue microenvironment. Adipogenic assays revealed higher percentage of lipid accumulation area in ASCs from superficial subcutaneous adipose tissue compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001). The high adipogenic potential of superficial subcutaneous adipose tissue was corroborated by an up-regulation of adipocyte fatty acid-binding protein (FABP4) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) and of C/EBPα (CCAAT/enhancer-binding protein alpha) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) microenvironments. Curiously, ASCs from retinacula cutis superficialis showed a higher level of adiponectin receptor gene compared with superficial subcutaneous adipose tissue (p = 0.0409), widely known as an anti-inflammatory hormone. Non-induced ASCs from retinacula cutis superficialis showed higher secretion of human vascular endothelial growth factor (VEGF), compared with superficial (p = 0.0485) and deep (p = 0.0112) subcutaneous adipose tissue and with adipogenic-induced ASCs from superficial (p = 0.0175) and deep (p = 0.0328) subcutaneous adipose tissue. Furthermore, ASCs from retinacula cutis superficialis showed higher secretion of Chemokine (C–C motif) ligand 5 (CCL5) compared with non-induced (p = 0.0029) and induced (p = 0.0089) superficial subcutaneous adipose tissue.ConclusionsThis study highlights the contribution to ASCs from retinacula cutis superficialis in their angiogenic property previously described for the whole superficial subcutaneous adipose tissue besides supporting its adipogenic potential for superficial subcutaneous adipose tissue.

Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.

Enhanced Neovascularization Using Injectable and rhVEGF‐Releasing Cryogel Microparticles

Cryogels are gel networks or scaffolds with a large porous structure; they can be tailored for injectability and for possessing a shape-memory ability. Herein, a growth factor-releasing cryogel microparticle (CMP) system is fabricated, and the therapeutic efficacy of recombinant human vascular endothelial growth factor (rhVEGF)-loaded CMP (V-CMP) for neovascularization is investigated. To prepare the cryogels, both methacrylated chitosan (Chi-MA) and methacrylated chondroitin sulfate (CS-MA) are used, and crosslinking using a radical crosslinking reaction is established. The physical, mechanical, and biological properties of the cryogels are analyzed by varying the amount of CS-MA used. The cryogels are then pulverized, and microsized CMPs are fabricated. CMPs dispersed in saline demonstrate a shear-thinning property, and can thus be extruded through a 23G needle. Additionally, V-CMP exhibit a sustained release profile of rhVEGF and enhance the in vitro proliferation of endothelial cells. Finally, neovascularization and effective tissue necrosis prevention are observed when V-CMPs are injected into a hindlimb ischemia mouse model. Thus, the injectable V-CMP system developed herein demonstrates a high potential utility in various tissue regeneration applications based on cell or growth factor delivery.