Expression Of Pro-angiogenic Factors Research Articles

Abstract 6817: Control of breast cancer via pro-resolving mediators

Abstract Tumor recurrence is the principal cause of mortality in breast cancer, yet the underlying mechanisms are not well understood. Anti-estrogen therapy, including tamoxifen, reduces tumor burden by inducing apoptosis and necrosis of cancerous tissue. The resulting cellular debris may stimulate tumor growth by disrupting inflammation resolution and creating pro-inflammatory and pro-tumorigenic microenvironments. Compared to estrogen receptor (ER)-positive breast cancer, ER-negative behaves aggressively, and patients have a poor prognosis due to the lack of treatment options. Here, we demonstrate that breast tumor cells killed by cytotoxic anti-estrogen therapy or chemotherapy (“tumor cell debris”) stimulate primary tumor growth when co-injected with a subthreshold (nontumorigenic) inoculum of tumor cells by triggering a macrophage-derived pro-inflammatory and pro-angiogenic “cytokine storm.” Thus, tumor cell debris is a critical risk factor for aggressive breast cancer growth. To assess whether stimulating the clearance of debris would impact breast cancer progression in vivo, we utilized maresin 1 (MaR1), maresin conjugates in tissue regeneration (MCTR1, MCTR2) and protectin conjugates in tissue regeneration (PCTRs), which are specialized pro-resolving lipid autacoid mediators (SPMs). We treated established models of ER-positive (EO771) and negative (4T1) breast cancer. Each maresin sharply reduced tumor growth in debris-stimulated and spontaneous (e.g. MMTV-PyMT) breast cancer models at nanogram concentrations without toxicity. Notably, PCTR1 inhibited orthotopic triple-negative (4T1) growth and proliferation compared to chemotherapy (paclitaxel) or immunotherapy (anti-cytotoxic T-lymphocyte-associated protein 4, CTLA4). Similarly, PCTR1 alone or in combination with chemotherapy (paclitaxel) reduced gene expression and protein levels of pro-angiogenic factor CXCL12/SDF1 in the ER-negative (4T1) tumor microenvironment. In ER-positive (EO771), a triple combination of PCTR1, chemotherapy (paclitaxel), and immunotherapy (anti-CTLA4) treatment potentiated the immune checkpoint blockade. Maresins also stimulated macrophage phagocytosis of therapy-generated breast tumor cell debris, inhibited tumor angiogenesis, and dampened a therapy-induced cytokine storm, including TNF-α, MIP-2/CXCL2, CCL2/MCP-1, IL- 1ra/IL-1F3, and G-CSF. Expression of SPM receptors (e.g. resolvin D1 (RvD1) receptor ALX/FPR2, resolvin D2 (RvD2) receptor GPR18, maresin 1 (MaR1) receptor LGR6, and resolvin E1 (RvE1) receptor ChemR23) were specifically identified in breast cancer tissue. Taken together, the maresin and protectin pathway mediators may represent a new therapeutic approach to stimulate the resolution of inflammation in breast cancer. Citation Format: Sarina A. Virani, Michael Gillespie, Haixia Yang, Diane R. Bielenberg, Mark W. Kieran, Sui Huang, Charles N. Serhan, Dipak Panigrahy. Control of breast cancer via pro-resolving mediators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6817.

AAV-mediated VEGFA overexpression promotes angiogenesis and recovery of locomotor function following spinal cord injury via PI3K/Akt signaling

Spinal cord injury (SCI) is a disorder of the central nervous system resulting from various factors such as trauma, inflammation, tumors, and other etiologies. This condition leads to impairment in motor, sensory, and autonomic functions below the level of injury. Limitations of current therapeutic approaches prompt an investigation into therapeutic angiogenesis through persistent local expression of proangiogenic factors. Here, we investigated whether overexpression of adeno-associated virus (AAV)-mediated vascular endothelial growth factor A (VEGFA) in mouse SCI promoted locomotor function recovery, and whether the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was mechanistically involved. Three weeks before SCI, AAV-VEGFA was injected at the T10 level to induce VEGFA overexpression. Neurofunctional, histological, and biochemical assessments were done to determine tissue damage and/or recovery of neuromuscular and behavioral impairments. Daily injections of the PI3K/Akt pathway inhibitor LY294002 were made to assess a possible mechanism. AAV-VEGFA overexpression dramatically improved locomotor function and ameliorated pathological injury caused by SCI. Improved motor-evoked potentials in hindlimbs and more spinal CD31-positive microvessels were observed in AAV-VEGFA-overexpressing mice. LY294002 reduced PI3K and Akt phosphorylation levels and attenuated AAV-VEGFA-related improvements. In conclusion, sustained local AAV-mediated VEGFA overexpression in spinal cord can significantly promote angiogenesis and ameliorate locomotor impairment after SCI in a contusion mouse model through activation of the PI3K/Akt signaling pathway.

Turmocin Plus Suppresses Vascular Endothelial Growth Factor (VEGF) and Macrophage Infiltration in the Management of Perineal Wounds, Anal Fistula, Acute Anal Fissures and Haemorrhoids

Anorectal problems such as anal fistula, Crohn’s disease, haemorrhoids, and fissures are prevalent across the general population. Severe discomfort, inflammation, swelling, itching, and bleeding during defecation are common symptoms of anorectal disorders. Depending on the severity of the condition, several medical therapies or surgical procedures may be used to treat these diseases. Surgical treatments like fistulectomy and sphincterotomy or haemorrhoidectomy are highly intrusive and have a risk of recurrence. Furthermore, surgical procedures cause pain, inflammation, and perineal sores. These will lead to severe socio-economic ramifications in the patient’s life. Therefore, treatment options that aid in the reduction of inflammation, pain, and perineal wounds are critical for anorectal disease management. Herbal formulations that comprise turmeric (Curcuma longa) extract have anti-inflammatory, pain-relieving, and wound-healing properties. The purpose of the current study was to elucidate the effect of Turmocin Plus on the infiltration of inflammatory cells and the expression of pro-angiogenic factors in anorectal and lower gastrointestinal disorders. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and wound migration assays were performed to determine the results of Turmocin Plus on the viability and migration of inflammatory cells. The effect of Turmocin Plus on pro-angiogenic factors was determined using Western blot analysis and immunofluorescence. Further, we validate our in vitro findings in human fistula specimens using IHC. The investigation showed that Turmocin Plus inhibits immunological (RAW 264.7) cell migration while maintaining their viability. Inflammation and increased levels of Vascular Endothelial Growth Factor (VEGF) were observed in Inflammatory Bowel Disease (IBD), fistula, fissures, and higher-grade haemorrhoids. However, Turmocin Plus suppresses the VEGF expression in macrophages (RAW 264.7) cells. Furthermore, compared to untreated human fistula tissues, decreased expression of VEGF was observed in Turmocin Plus treated patient samples, validating the in vitro findings. Our study suggests that Turmocin Plus is a potent therapeutic formulation in treating fistula, perineal wounds, and Crohn’s disease.

Roles of endothelial cell specific molecule‑1 in tumor angiogenesis (Review).

Angiogenesis plays a crucial role in tumor growth and metastasis, and is heavily influenced by the tumor microenvironment (TME). Endothelial cell dysfunction is a key factor in tumor angiogenesis and is characterized by the aberrant expression of pro-angiogenic factors. Endothelial cell specific molecule-1 (ESM1), also known as endocan, is a marker of endothelial cell dysfunction. Although ESM1 is primarily expressed in normal endothelial cells, dysregulated ESM1 expression has been observed in human tumors and animal tumor models, and implicated in tumor growth, metastasis and angiogenesis. The precise role of ESM1 in tumor angiogenesis and its potential regulatory mechanisms are not yet conclusively defined. However, the aim of the present review was to explore the involvement of ESM1 in the process of tumor angiogenesis in the TME and the characteristics of neovascularization. In addition, the present review discusses the interaction between ESM1 and angiogenic factors, as well as the mechanisms through which ESM1 contributes to tumor angiogenesis. Furthermore, the reciprocal regulation between ESM1 and the TME is explored. Finally, the potential of targeting ESM1 as a therapeutic strategy for tumor angiogenesis is presented.

Abstract A040: Assessing the ability of Fc3TSR to remodel the tumor microenvironment and enhance efficacy of immunotherapies and chemotherapy in a murine model of pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) has a poor survival rate due to late diagnosis where metastasis has often occurred. Angiogenesis, the process by which new vessels form from pre-existing vasculature, is crucial for tumor growth and metastasis. Tumors aggressively upregulate expression of pro-angiogenic factors, stimulating rapid vessel formation. These vessels often lack pericyte coverage and have dysfunctional morphology, leading to high interstitial fluid pressure (IFP). Impaired perfusion and high IFP impede therapy uptake. Fc3TSR is a novel fusion protein derived from the potent angiogenic inhibitor thrombospondin 1, which we have shown to normalize vasculature in ovarian cancer, significantly decreasing tumor size and improving uptake of many therapies. In this study, we evaluated Fc3TSR’s ability to remodel the tumor microenvironment to induce tumor regression and to enhance efficacy of other therapies. Methods: We developed an orthotopic syngeneic murine model of PDAC, whereby we surgically injected 2.5 × 104 murine PDAC cells (KPC) into the tail of the pancreas of C57BL/6 mice. Tumors were allowed to progress for 14 days before intraperitoneal (IP) administration of Fc3TSR (0.158mg/kg) or PBS (control) on day 14 and 21. Gemcitabine (GEM) chemotherapy treated mice received either daily, metronomic dosages of GEM, or weekly, maximum tolerated dosages GEM starting on day 23. Checkpoint inhibitor mice received either PD-L1 (25ug) or CTLA4 (25ug) checkpoint inhibitors on day 23 and 26. Mice were euthanized on day 30 and tumors and the draining lymph nodes were collected and weighed. 1 hour before euthanasia, Fc3TSR and PBS mice were IP injected with Hypoxyprobe (Pimonidazole Hydrochloride) and tumors were immunostained for markers of blood vessels and tumor hypoxia. Results: Fc3TSR induced tumor regression when compared to PBS, but GEM at either dosage did not further enhance this effect. Following Fc3TSR, PD-L1, but not CTLA-4 checkpoint inhibitors significantly reduced tumor size when compared to mice pre-treated with PBS. Fc3TSR treatment also reduced the area of tumor hypoxia. Tumor vasculature is currently being imaged and analyzed for Fc3TSR’s effect on vessel morphology. Conclusion: Our data suggests that normalizing the tumor microenvironment can enhance the uptake and efficacy of combination therapies. Enhanced perfusion could facilitate the migration of activated immune cells and may enhance immune responses in PDAC patients. Here we demonstrate a novel approach to optimize therapeutic efficacy in advanced stage PDAC. Citation Format: Bianca Garlisi, Caroline Aitken, Sylvia Lauks, Julia Stewart, Duncan Petrik, Jack Lawler, Jim Petrik. Assessing the ability of Fc3TSR to remodel the tumor microenvironment and enhance efficacy of immunotherapies and chemotherapy in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A040.

Buyang Huanwu Decoction promotes neurovascular remodeling by modulating astrocyte and microglia polarization in ischemic stroke rats

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHWD), one of the most commonly utilized traditional Chinese medicine prescription for treatment of cerebral ischemic stroke. However, the understanding of BYHWD on neurovascular repair following cerebral ischemia is so far limited. Aim of the studyThis research investigated the influence of BYHWD on neurovascular remodeling by magnetic resonance imaging (MRI) technology and revealed the potential neurovascular repair mechanism underlying post-treatment with BYHWD after ischemic stroke. Materials and methodsMale Sprague–Dawley rats were utilized as an ischemic stroke model by permanent occlusion of the middle cerebral artery (MCAO). BYHWD was intragastrically administrated once daily for 30 days straight. Multimodal MRI was performed to detect brain tissue injuries, axonal microstructural damages, cerebral blood flow and intracranial vessels on the 30th day after BYHWD treatment. Proangiogenic factors, axonal/synaptic plasticity-related factors, energy transporters and adenosine monophosphate-activated protein kinase (AMPK) signal pathway were evaluated using western blot. Double immunofluorescent staining and western blot were applied to evaluate astrocytes and microglia polarization. ResultsAdministration of BYHWD significantly alleviated infarct volume and brain tissue injuries and ameliorated microstructural damages, accompanied with improved axonal/synaptic plasticity-related factors, axonal growth guidance factors and decreased axonal growth inhibitors. Meanwhile, BYHWD remarkably improved cerebral blood flow, cerebral vascular signal and promoted the expression of proangiogenic factors. Particularly, treatment with BYHWD obviously suppressed astrocytes A1 and microglia M1 polarization accompanied with promoted astrocyte A2 and microglia M2 polarization. Furthermore, BYHWD effectively improved energy transporters. Especially, BYHWD markedly increased expression of phosphorylated AMPK, cyclic AMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) accompanied by inactivation of the NF-κB. ConclusionTaken together, these findings identified that the beneficial roles of BYHWD on neurovascular remodeling were related to AMPK pathways -mediated energy transporters and NFκB/CREB pathways.

Ang1 immunoexpression vs vascular profile in chorio-villous germinative status in early term compromised pregnancies

Background: A normal maternal-fetal system is essential for the functioning of the placenta during placentation and the establishment of the maternalembryofetal vascular circulation. The angiopoietin/TIE pathway is involved in vascular morphogenesis through regulation, survival and maturation of endothelial cells concomitant with vascular remodeling. Deregulation of pro-angiogenic factor secretion and expression is associated with disruption of vascular morphogenesis, reduction of vascular bed and installation of primary placental insufficiency. The aim: Evaluation of Ang 1 immunoexpression in early term compromised pregnancies in the context of chorio-villary circulatory dysfunction in primary placental insufficiency. Material and methods: Abortion product from 61 patients (stagnant pregnancies – 39 cases, early miscarriage – 8 cases, control group – 14 cases of pregnancies solved on social indications/ desire) were immunohistochemically evaluated with the marker for anti-Ang1 and anti-CD31. Results: The villous syncytiotrophoblast was the most immunoreactive area. Most of cases of the pregnancies terminated on social indications/ desire were anti-Ang1 negative. The levels of anti-Ang1 immunoexpression were statistically significantly different in case of syncytiotrophoblast of early miscarriages and abortions terminated on social indications/ desire. The highest chorio-villous vascular density was noticed in the abortions on social indications/ desire and early misscariages group. Conclusions: The placental period is characterized by a weak angiogenic Ang1 differentiated cellular environment in the chorio-villous germinal site in the group of short term compromised pregnancies. The selectively immunoexpressed cellular profile statistically significantly correlates with placental vascular index and chorio-villous vascular density in stagnant pregnancies.

Ang2 immunoexpression vs vascular profile in chorio-villous germinative status in early term compromised pregnancies

Background: The angiopoietin/TIE system is one of the signalling pathways that regulate vascular development and remodelling during morphogenesis of the placental complex. Deregulation of the vascularization process is associated with primary placental insufficiency through destabilization of the maternal-fetal functional system. Disruption of expression of pro-angiogenic factors is associated with reduction of the microcirculatory bed and installation of circulatory dysfunction. The aim: Evaluation of Ang2 immunoexpression in early term compromised uterine pregnancies in the context of chorio-villous circulatory dysfunction in primary placental insufficiency. Material and methods: Abortion product from 67 patients with early term compromised pregnancies (stagnant pregnancies – 43 cases, early miscarriages – 11 cases, control batch that included 13 cases of pregnancies solved on social indications) were immunohistochemically evaluated with the marker for anti-Ang2 and anti-CD31. Results: The majority of sites analyzed were Ang2 negative, with the exception of syncytiotrophoblast, that reached the highest score (+3) in most cases. A maximum placental vascularisation index was achieved in the control group. The stagnant pregnacies batch has registered the lowest vascular density mean. Conclusions: The placentation period is characterized by a weak Ang2 cellular environment in the chorio-villous germinal site in the group of short-term compromised pregnancies, except a highly positive syncytiotrophoblast. The immunoexpression profile in vascular endothelium correlates statistically significantly with placental vascularization index and chorio-villous vascular density in stagnant pregnancies.

Blood flow diverts extracellular vesicles from endothelial degradative compartments to promote angiogenesis.

Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We demonstrated that efficient uptake of tEVs occurs in venous endothelial cells that are subjected to hemodynamics. Low flow regimes observed in veins partially reroute internalized tEVs toward non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in low flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that low flow regimes potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics.

Regulation of BACH1 expression by hemin improves cardiac function and revascularisation in a mouse model of myocardial infarction

Abstract Therapies to induce mature neovascularization formation and moderate oxidative stress after myocardial infarction (MI) are urgently needed. The BACH1 transcription factor is known as a repressor of genes involved in the antioxidant response (1). We have demonstrated BACH1 can regulate neovascularization through modulation of pro-angiogenic genes, particularly angiopoietin-1 (2). We hypothesise expression of BACH1 after MI contributes to suppression of genes which promote reparative angiogenesis. We propose treatment with hemin (a therapeutic compound known to degrade BACH1) will prevent BACH1 repression of target genes, leading to increased expression of proangiogenic factors and increased neovascularisation and cardiac repair in a mouse MI model. MI was induced in 8-week old C57/Bl6 male mice by permanent ligation of the left anterior descending coronary artery, n=10-14. Expression of Bach1 and the antioxidant Hmox1 in heart tissue was determined by Western blot at day 3 and 10 post MI, whilst a second group were randomized to receive vehicle, 12.5, 25 or 50mg/kg hemin intraperitoneally every 3 days until 28 days post-MI. On day 28, following echocardiography, mice were euthanised and ventricular tissue collected for histological analysis. Data was analysed by 2-way ANOVA with post-test multiple comparisons. Compared to sham mice, Bach1 expression significantly increased at both day 3 and 10 post MI (P<0.0001 and P<0.01 respectively). Bach1 expression at day 10 was significantly lower compared to day 3, suggesting a transient effect (P<0.01). Hmox1 expression was significantly decreased at both timepoints (both P<0.001 vs. sham) suggesting in the initial period after MI the antioxidant and angiogenic responses are suboptimal. Treatment of mice with 25mg/kg hemin for 28 days post MI caused significant increases in capillary and arteriole density in the peri-infarct zone (P<0.0002 and P<0.0008 vs. vehicle respectively). There was no effect on capillary or arteriole density in the remote zone. In vehicle-treated mice there was thinning of the left ventricular end-diastolic anterior wall thickness (LVAW) corresponding to the infarct area. Interestingly, a significant increase in the thickness of the LVAW was observed after 25mg/kg hemin treatment (P<0.0001 vs. vehicle), whilst LV internal diameter significantly decreased (P<0.0001 vs. vehicle). Ejection fraction (P<0.0001), fractional shortening (P<0.0009) and stroke volume (P<0.0001) all significantly increased in mice treated with hemin compared to vehicle. Treatment of mice with 25mg/kg hemin for 28 days post MI was sufficient to induce significantly increased capillary and arteriole density in the peri-infarct region. We also observed hemin exerted benefits on volumetric and contractility indexes. This data suggests hemin has potential to be used therapeutically post MI to improve revascularisation and cardiac function.

Clickable corneal neovascularization therapy with ROS-responsive polydopamine silica nanoparticles loaded with fenofibrate

Corneal neovascularization is a severe eye disease that is often associated with an inflammatory cycle and increased oxidative stress. Excessive reactive oxygen species can cause harmful changes in the corneal epithelium and lead to vicious inflammatory cycles, resulting in an increase in corneal neovascularization. Unfortunately, many drugs used in clinic are water-insoluble and have low bioavailability on the ocular surface, which limits their effectiveness. Therefore, it's crucial to design a new drug delivery system. In this study, we designed an oxide-responsive nanoparticle system called MPA, which was consisted of the large pore sized-mesoporous silica nanoparticles (MSNs) core and the polydopamine (PDA) surface shell via the linkage with disulfide bond. Finally, the nanoparticles are endowed with the enhanced loading efficiency and the controllable release of fenofibrate (Feno) in response to the abundant ROS level in the inflammatory environment. In an in vivo study using mice, treatment with MPA@ Feno resulted in minimal corneal neovascularization area and length and significantly reduced expression of pro-angiogenic factors and oxidative stress factors. Therefore, MPA solves the problem of poor drug solubility, clearing oxygen species in the pathological environment, which will improve its therapeutic effect in corneal neovascularization.

3D printed porous PLGA/n-HA/MgP composite scaffolds with improved osteogenic and angiogenic properties

The osteogenic and angiogenic characteristics are crucial for the selection of bone substitute materials and structural design. This study proposed a porous PLGA/n-HA/MgP composited scaffold by printing of the poly (lactide-coglycolide) (PLGA), nano-hydroxyapatite (n-HA), and magnesium phosphate (MgP). The physicochemical properties, osteogenic activities, and pro-angiogenic impacts of porous 3D printed PLGA/n-HA scaffolds with variable MgP concentration were investigated. The results indicated that the presence of MgP in the composite promoted scaffold degradation and buffered the acidic environment induced by PLGA degradation. The highest pro-osteogenic activity has been observed in PLGA/n-HA/10% MgP (PH10M) group, which up-regulated BMP2 and RUNX2 factors in vitro and in vivo. Nevertheless, at a maximal ion release of 7.2 mmol/L, PLGA/n-HA/30% MgP (PH30M) marginally reduced theosteogenic differentiation of osteoblasts through the RANKL/OPG pathway. In vitro, PLGA/n-HA/20% MgP (PH20M) demonstrated superior migration, pro-angiogenic factor expression, and angiogenesis compared to other groups, and the histological investigation corroborated the improved angiogenesis in PH10M and PH20M. In conclusion, suitable MgP ratio shows the potential to improve the osteogenic and angiogenic properties of PLGA/n-HA-based composites in orthopedic applications.

The Inhibition of the FGFR/PI3K/Akt Axis by AZD4547 Disrupts the Proangiogenic Microenvironment and Vasculogenic Mimicry Arising from the Interplay between Endothelial and Triple-Negative Breast Cancer Cells.

Vasculogenic mimicry (VM), a process in which aggressive cancer cells form tube-like structures, plays a crucial role in providing nutrients and escape routes. Highly plastic tumor cells, such as those with the triple-negative breast cancer (TNBC) phenotype, can develop VM. However, little is known about the interplay between the cellular components of the tumor microenvironment and TNBC cells' VM capacity. In this study, we analyzed the ability of endothelial and stromal cells to induce VM when interacting with TNBC cells and analyzed the involvement of the FGFR/PI3K/Akt pathway in this process. VM was corroborated using fluorescently labeled TNBC cells. Only endothelial cells triggered VM formation, suggesting a predominant role of paracrine/juxtacrine factors from an endothelial origin in VM development. Via immunocytochemistry, qPCR, and secretome analyses, we determined an increased expression of proangiogenic factors as well as stemness markers in VM-forming cancer cells. Similarly, endothelial cells primed by TNBC cells showed an upregulation of proangiogenic molecules, including FGF, VEGFA, and several inflammatory cytokines. Endothelium-dependent TNBC-VM formation was prevented by AZD4547 or LY294002, strongly suggesting the involvement of the FGFR/PI3K/Akt axis in this process. Given that VM is associated with poor clinical prognosis, targeting FGFR/PI3K/Akt pharmacologically may hold promise for treating and preventing VM in TNBC tumors.

MDMB-FUBINACA Influences Brain Angiogenesis and the Expression of VEGF, ANG-1, and ANG-2.

This study aims to explore the impact of the synthetic cannabinoid methyl 2-(1-(4- fluorobenzyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (MDMB-FUBINACA) on the angiogenesis process in human brain microvascular endothelial cells. Synthetic cannabinoids (SCs) are substances that mimic the natural components found in the cannabis plant. SCs are considered prohibited substances that have a clear impact on the central nervous system (CNS). The purpose of this study is to explore how MDMB-FUBINACA influences angiogenesis in human brain microvascular endothelial cells and to clarify the pathways related to the cannabinoid receptors. Human brain microvascular endothelial cells (hBMECs) were grown in the medium containing Dulbecco Modified Eagle Medium (DMEM/F12) using an endothelial cell growth kit. Endothelial cell viability was evaluated using the MTT test. Migration ability was measured using the Wound healing test. The angiogenic capability was measured using a Tube Formation assay. Real-time polymerase chain reaction (RT-PCR) was utilized to explore the mRNA concentrations following MDMBFUBINACA treatment. ELISA and Western blotting were also employed to measure the protein levels. MDMB-FUBINACA greatly increases tube formation, endothelial cell proliferation, and migration. Pro-angiogenic factors such as angiopoietins 1 and 2 (ANG-1 and 2) and vascular endothelial growth factor (VEGF) were shown to be increased at both the RNA and protein levels. MDMB-FUBINACA induces the progression of the angiogenesis process by inducing the expression of pro-angiogenic factors. These findings aim toward developing novel treatments for angiogenesis- related disorders.

In Vitro Coculture of Primary Human Cells to Analyze Angiogenesis, Osteogenesis, and the Inflammatory Response to Newly Developed Osteosynthesis Material for Pediatric Maxillofacial Traumatology: A Potential Pretesting Model before In Vivo Experiments

During the present study, an in vitro coculture bone tissue mimic based on primary osteoblasts and primary endothelial cells was used for a complex and broad evaluation of a newly developed material for applications in pediatric maxillofacial traumatology. The biomaterial was composed of PDLLA (poly(D,L-lactide)) in various combinations with calcium carbonate (CC), magnesium (Mg), and chitosan (CH). Besides classical biocompatibility analyses, the present study evaluated material-dependent effects on fundamental processes that are essential for successful material integration and regeneration. Therefore, inflammation-associated factors such as E-selectin and interleukins were analyzed in the in vitro model system on gene expression and protein level depending on the different materials. Furthermore, in order to test the capability of vascularization of the material, the effect of the different materials on the formation of microvessel-like structures as well as the expression and release of proangiogenic factors was investigated in vitro in the bone coculture model. In addition, the mineralization capacity as well as the relative gene expression of osteogenic differentiation factors was analyzed in response to the different materials. As a result, the authors could assess the material combination PDLLA: CC CH as the most functionally tested material with regard to biocompatibility, inflammatory response, and microvessel-like structure formation as well as osteogenic differentiation in the in vitro coculture system. In conclusion, by using tissue-engineered human bone tissue equivalents as proposed here in an in vitro coculture model, biomaterial-mediated effects can be readily investigated. Moreover, it is proposed that these complex in vitro evaluations could contribute to the understanding and improvement of the development of novel materials for pediatric traumatological care for prospective clinical applications.

An Engineered Dermal Substitute with Mesenchymal Stem Cells Enhances Cutaneous Wound Healing.

The treatment of refractory cutaneous wounds remains to be a clinical challenge. There is growing evidence to show that mesenchymal stem cells (MSCs) have great potential in promoting wound healing. However, the therapeutic effects of MSCs are greatly dampened by their poor survival and engraftment in the wounds. To address this limitation, in this study, MSCs were grown into a collagen-glycosaminoglycan (C-GAG) matrix to form a dermis-like tissue sheet, named engineered dermal substitute (EDS). When seeded on C-GAG matrix, MSCs adhered rapidly, migrated into the pores, and proliferated readily. When applied onto excisional wounds in healthy and diabetic mice, the EDS survived well, and accelerated wound closure, compared with C-GAG matrix alone or MSCs in collagen hydrogel. Histological analysis revealed that EDS prolonged the retention of MSCs in the wounds, associated with increased macrophage infiltration and enhanced angiogenesis. RNA-Seq analysis of EDS-treated wounds uncovered the expression of abundant human chemokines and proangiogenic factors and their corresponding murine receptors, suggesting a mechanism of ligand/receptor-mediated signals in wound healing. Thus, our results indicate that EDS prolongs the survival and retention of MSCs in the wounds and enhances wound healing.

Adipose mesenchymal stem cell-derived soluble factors, produced under hypoxic condition, efficiently support in vivo angiogenesis

Tissue regeneration or healing both require efficient vascularization within a tissue-damaged area. Based on this concept, a remarkable number of strategies, aimed at developing new tools to support re-vascularization of damaged tissue have emerged. Among the strategies proposed, the use of pro-angiogenic soluble factors, as a cell-free tool, appears as a promising approach, able to overcome the issues concerning the direct use of cells for regenerative medicine therapy. Here, we compared the effectiveness of adipose mesenchymal stem cells (ASCs), use as cell suspension, ASC protein extract or ASC-conditioned-medium (i.e., soluble factors), combined with collagenic scaffold, in supporting in vivo angiogenesis. We also tested the capability of hypoxia in increasing the efficiency of ASC to promote angiogenesis, via soluble factors, both in vivo and in vitro. In vivo studies were performed using the Integra® Flowable Wound Matrix, and the Ultimatrix in sponge assay. Flow cytometry was used to characterize the scaffold- and sponge-infiltrating cells. Real-time PCR was used to evaluate the expression of pro-angiogenic factors by stimulating Human Umbilical-Vein Endothelial Cells with ASC-conditioned media, obtained in hypoxic and normoxic conditions. We found that, in vivo, ACS-conditioned media can support angiogenesis similar to ASCs and ASC protein extract. Also, we observed that hypoxia increases the pro-angiogenic activities of ASC-conditioned media, compared to normoxia, by generating a secretome enriched in pro-angiogenic soluble factors, with bFGF, Adiponectine, ENA78, GRO, GRO-a, and ICAM1-3, as most regulated factors. Finally, ASC-conditioned media, produced in hypoxic condition, induce the expression of pro-angiogenic molecules in HUVECs. Our results provide evidence that ASC-conditioned-medium can be proposed as a cell-free preparation able to support angiogenesis, thus providing a relevant tool to overcome the issues and restrictions associated with the use of cells.

Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia

Objective: Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways. Methods: Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium. Results: Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p <0.001) and matrix metalloprotease-2 (MMP-2, p <0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p >0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged. Conclusions: In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.

Photoactivated MXene Nanosheets for Integrated Bone-Soft Tissue Therapy: Effect and Potential Mechanism.

The bone defects caused by trauma are inevitably accompanied by soft tissue damage. The development of multifunctional bioactive biomaterials with integrated bone and soft tissue regeneration is necessary and needed urgently in orthopedics. In this work, we found that the photoactivated MXene (Ti3C2Tx) nanosheet showed positive effects on promoting both bone and soft tissue regeneration. We further investigated the detailed effect and potential mechanism of photoactivated MXene on tissue regeneration. Photoactivated MXene shows a good thermal effect and robust antibacterial activity to inhibit the expression of inflammation factors and methicillin-resistant Staphylococcus aureus (MRSA) infection and induces the expression of pro-angiogenic factors and soft tissue wound repair. Photoactivated MXene can also regulate the osteogenic differentiation of adipose-derived stem cells (ADSCs) through the ERK signaling pathway by activating the heat shock protein 70 (HSP70) and enhancing the repair of bone tissue. This work sheds light on the development of bioactive MXene with photothermal activation as an efficient strategy for bone and soft tissue regeneration simultaneously.

Targeted dexamethasone nano-prodrug for corneal neovascularization management

BackgroundTo overcome the drawbacks of traditional therapy for corneal neovascularization (CNV), we evaluated the efficacy of polyethylene glycol (PEG)-conjugated Ala-Pro-Arg-Pro-Gly (APRPG) peptide modified dexamethasone (Dex), a novel nano-prodrug (Dex-PEG-APRPG, DPA). MethodsCharacterization of DPA nano-prodrug were measured with transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Cytotoxicity and effects on cell migration and tube formation of DPA were evaluated in vitro. A murine CNV model was established by cornea alkali burn. The injured corneas were given eye drops of DPA (0.2 mM), Dex solution (0.2 mM), Dexp (2 mM), or normal saline three times a day. After two weeks, eyes were obtained for the analysis of histopathology, immunostaining, and mRNA expression. ResultsDPA with an average diameter of 30 nm, presented little cytotoxicity and had good ocular biocompatibility. More importantly, DPA showed specific targeting to vascular endothelial cells with efficient inhibition on cell migration and tube formation. In a mouse CNV model, clinical, histological, and immunohistochemical examination results revealed DPA had a much stronger angiogenesis suppression than Dex, resembling a clinical drug with an order of magnitude higher concentration. This was ascribed to the significant downregulations in the expression of pro-angiogenic and pro-inflammatory factors in the corneas. In vivo imaging results also demonstrated that APRPG could prolong ocular retention time. ConclusionsThis study suggests that DPA nano-prodrug occupies advantages of specific targeting ability and improved bioavailability over conventional therapy, and holds great potential for safe and efficient CNV therapy.