Angiogenic Capacity Of Endothelial Cells Research Articles

IFITM3 promotes glioblastoma stem cell-mediated angiogenesis via regulating JAK/STAT3/bFGF signaling pathway

Interferon-induced transmembrane protein 3 (IFITM3) has been previously verified to be an endosomal protein that prevents viral infection. Recent findings suggested IFITM3 as a key factor in tumor invasion and progression. To clarify the role and molecular mechanism of IFITM3 in Glioblastoma multiforme (GBM) progression, we investigated the expression of IFITM3 in glioma datasets culled from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Primary GBM stem cells (GSCs) were cultured and identified in vitro. Loss-of-function and gain-of-function experiments were established by using shRNAs and lentiviral vectors targeting IFITM3. Co-culture system of GSCs and vascular endothelial cells was constructed in a Transwell chamber. Tube formation and spheroid-based angiogenesis assays were performed to determine the angiogenic capacity of endothelial cells. Results revealed that IFITM3 is elevated in GBM samples and predictive of adverse outcome. Mechanistically, GSCs-derived IFITM3 causes activation of Jak2/STAT3 signaling and leads to robust secretion of bFGF into tumor environment, which eventually results in enhanced angiogenesis. Taken together, these evidence indicated IFITM3 as an essential factor in GBM angiogenesis. Our findings provide a new insight into mechanism by which IFITM3 modulates GBM angiogenesis.

Characterization of Patient-Derived GNAQ Mutated Endothelial Cells from Capillary Malformations

Capillary malformations (port-wine stains) are congenital skin lesions that are characterized by dilated capillaries and post-capillary venules. Capillary malformations are caused by altered functioning of the vascular endothelium. Somatic genetic mutations have predominantly been identified in the endothelial cells of capillary malformations, providing an opportunity for the development of targeted therapies. However, there is currently limited in-depth mechanistic insight in the pathophysiology and a lack of pre-clinical research approaches. In a mono-centre exploratory study of 17 adult patients with capillary malformations, we found somatic mutations in the GNAQ [p.R183Q, p.R183G or p.Q209R] or GNA11 [p.R183C] genes. We applied an endothelial-selective cell isolation protocol to culture primary endothelial cells from skin biopsies from these patients. In 3 out of the 17 cases we succesfully expanded patient-derived cells in culture, while maintaining endothelial specificity as demonstrated by Vascular Endothelial (VE)-cadherin immunostainings. In addition, we tested the angiogenic capacity of endothelial cells from a patient with a GNAQ [p.R183G]. These proof-of-principle results reveal that primary cells isolated from capillary malformations may represent a functional research model to investigate the role of endothelial somatic mutations in the aetiology of capillary malformations, but improved isolation and culture methodologies are urgently needed to advance the field.

Comparative analysis of single-cell transcriptome reveals heterogeneity in the tumor microenvironment of lung adenocarcinoma and brain metastases

PurposeSolid tumors such as lung adenocarcinoma include not only the tumor cells but also the microenvironment in which the tumor cells continuously interact with each other. An in-depth understanding of the oncological features and tumor microenvironment (TME) of lung adenocarcinoma and brain metastases at the single-cell level could provide new therapeutic strategies for brain metastases from lung adenocarcinoma.MethodsTo solve this problem, we performed single-cell RNA sequencing (scRNA-seq) analysis on 15 lung adenocarcinoma samples and 10 brain metastasis samples.ResultsA total of 86,282 single cells were obtained and divided into 8 cell types, including epithelial cells, endothelial cells, fibroblasts, oligodendrocytes, T/NK cells, B cells, mast cells, and macrophages. In brain metastases, we found a significantly lower proportion of T/NK cells and mast cells, and more severe immune dysregulation. In addition, we found a subpopulation of macrophages with high expression of metastasis-promoting-related genes enriched in brain metastatic tissues. Moreover, in brain metastases, we found a significantly increased proportion of myofibroblastic cancer-associated fibroblasts (myCAFs) and a higher angiogenic capacity of endothelial cells. Epithelial cells in brain metastases were more malignant and underwent genomic reprogramming. Next, we found that DNA damage-inducible transcript 4 (DDIT4) expression was upregulated in epithelial cells in brain metastases and was associated with poor prognosis. Finally, we experimentally validated that the downregulation of DDIT4 inhibited the proliferation, migration, and invasion of lung cancer cells.ConclusionsThis study depicts a single-cell atlas of lung adenocarcinoma and brain metastases by scRNA-seq and paves the way for the development of future therapeutic targets for brain metastases from lung cancer.

Lignin/Puerarin Nanoparticle-Incorporated Hydrogel Improves Angiogenesis through Puerarin-Induced Autophagy Activation.

Puerarin is the main isoflavone extracted from Radix Puerariae lobata (Willd.) and exerts a strong protective effect on endothelial cells. This isoflavone also exerts proven angiogenic effects; however, the potential underlying mechanism has not been fully explored. Here in this work, we aimed to determine the proangiogenesis effect of a puerarin-attached lignin nanoparticle-incorporated hydrogel and explore the underlying mechanism. Puerarin-attached lignin nanoparticles were fabricated and mixed with the GelMA hydrogel. After the hydrogel was characterized, the angiogenic effect was evaluated in a mouse hind-limb ischemia model. To further explore the mechanism of angiogenesis, human endothelial cell line EA.hy926 was exposure to different concentrations of puerarin. Wound healing assays and tube formation assays were used to investigate the effects of puerarin on cell migration and angiogenesis. qPCR and Western blotting were performed to determine the changes in the levels of angiogenesis indicators, autophagy indicators and PPARβ/δ. 3-MA was used to assess the role of autophagy in the puerarin-mediated angiogenesis effect in vivo and in vitro. The hydrogel significantly improved blood flow restoration in mice with hind-limb ischemia. This effect was mainly due to puerarin-mediated increases in the angiogenic capacity of endothelial cells and the promotion of autophagy activation. A potential underlying mechanism might be that puerarin-mediated activation of autophagy could induce an increase in PPARβ/δ expression. The puerarin-attached lignin nanoparticle-incorporated hydrogel effectively alleviated blood perfusion in mice with hind-limb ischemia. Puerarin has a prominent proangiogenic effect. The potential mechanisms might be that puerarin-mediated autophagy activation and increase in PPARβ/δ.

SP05. A Novel, Patient-specific, Organotypic 3D Platform for Modeling Cell Behavior in the Setting of Malignancy

PURPOSE: With the advent of personalized cancer treatments, patient-specific in vitro models of the breast malignancies are critical for modeling the complex in vivo milieu and to better assess therapeutic efficacy. A primary challenge to developing such platforms is isolating and successfully co-culturing the many primary cell types that constitute the tumor microenvironment. In addition, optimizing for cost-effectiveness of such platforms is critical to making the technology translational. Using patient-derived cells and machine learning-based image analysis, we have developed a low-cost 3D biomimetic platform that allows for the study of cell behavior in a highly organotypic model of breast malignancy. METHODS: Stromal vascular fraction, organoids and mature adipocytes were isolated from breast tissue obtained from healthy female patients undergoing reduction mammoplasty. Isolated cells were embedded in non-ribosylated collagen, creating a biomimetic milieu that approximates the patient-specific in vivo cellular environment (“biomimetic collagen”). 3D collagen constructs consisting of a bottom layer of plain collagen or collagen embedded with RFP-tagged MDA-231 tumor cells followed by a layer of plain or biomimetic collagen were built in a 96-well plate. GFP-tagged human umbilical vascular endothelial (HUVEC) cells were plated in a monolayer layer on top of the collagen to mimic the endothelial barrier. Constructs were imaged with confocal microscopy on days 0 and 7 to assess for endothelial cell organization and migration. Image analysis was completed in Imaris. ANOVA tests for statistical significance were completed in R Studio. RESULTS: Confocal microscopy at day 0 revealed ~800μm thick constructs with distinct tumor and non-tumor collagen layers and a robust HUVEC top monolayer. Confocal images at day 7 were notable organization of the HUVEC layer into 2D tubular networks with some HUVEC below the top monolater indicating migration through the collagen matrix. Our machine-learning based approach was able to reliably and reproducibly parse HUVEC cells from the surrounding cells and collagen, allowing for quantification of cell migration in the Z-axis. Statistical analysis revealed a significant difference in migration across treatment groups (p < 0.01), with endothelial cells in the tumor and biomimetic collagen platform migrating 20-40 microns further than endothelial cells in the other construct designs, a finding retained on intergroup pairwise comparison (p < 0.01). CONCLUSION: This biomimetic platform design and image analysis allows for reliable, reproducible study of discrete endothelial cell interactions with the tumor microenvironment. The finding of enhanced HUVEC migration in the platform with both tumor and biomimetic cells is consistent with the hypothesis that stromal cells, adipocytes and malignant cells enhance the angiogenic capacity of endothelial cells. Importantly, this utilization of patient-derived stromal vascular fraction and mature adipocytes in this platform makes it inherently personalized, a vital component of any future clinically-relevant in vitro cancer platform.

The Proangiogenic Potential of Rat Adipose-Derived Stromal Cells with and without Cell-Sheet Induction: A Comparative Study.

A functional vasculature for survival remains a challenge for tissue regeneration, which is indispensable for oxygen and nutrient supply. Utilizing mesenchymal stromal cells (MSCs) to alleviate tissue ischemia and repair dysfunctional or damaged endothelium is a promising strategy. Compared to other populations of MSCs, adipose-derived stromal cells (ASCs) possess a more significant proangiogenic potential and are abundantly available. Cell sheet technology has recently been widely utilized in bone engineering. Compared to conventional methods of seeding seed cell suspension onto biological scaffolds, cell sheet technology prevents cell loss and preserves the extracellular matrix (ECM). Nevertheless, the proangiogenic potential of ASC sheets remains unknown. In this study, rat ASC sheets were constructed, and their macro- and microstructures were examined. In addition, we investigated the effects of ASCs and ASC sheets on the biological properties and angiogenic capacity of endothelial cells (ECs). The results demonstrated that the ASC sheets gradually thickened as the number of cells and ECM increased over time and that the cells were in an active state of secretion. Similar to ASC-CM, the conditioned medium (CM) of ASC sheets could significantly enhance the proliferative capacity of ECs. ASC sheet-CM has significant advantages over ASC-CM in promoting the migration and angiogenesis of ECs, where the exosomes secreted by ASC sheets play an essential role. Therefore, using ASC sheets for therapeutic tissue and organ regeneration angiogenesis may be a valuable strategy.

A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

Background:TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium.Methods:We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo.Results:Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression.Conclusions:Our work uncovers a TP-driven COX-2–dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Protein tyrosine phosphatase 1B inhibition improves endoplasmic reticulum stress‑impaired endothelial cell angiogenic response: A critical role for cell survival.

Endoplasmic reticulum (ER) stress contributes to endothelial dysfunction, which is the initial step in atherogenesis. Blockade of protein tyrosine phosphatase (PTP)1B, a negative regulator of insulin receptors that is critically located on the surface of ER membrane, has been found to improve endothelial dysfunction. However, the role of ER stress and its related apoptotic sub‑pathways in PTP1B‑mediated endothelial dysfunction, particularly its angiogenic capacity, have not yet been fully elucidated. Thus, the present study aimed to investigate the impact of PTP1B suppression on ER stress‑mediated impaired angiogenesis and examined the contribution of apoptotic signals in this process. Endothelial cells were exposed to pharmacological ER stressors, including thapsigargin (TG) or 1,4‑dithiothreitol (DTT), in the presence or absence of a PTP1B inhibitor or small interfering (si)RNA duplexes. Then, ER stress, angiogenic capacity, cell cycle, apoptosis and the activation of key apoptotic signals were assessed. It was identified that the inhibition of PTP1B prevented ER stress caused by DTT and TG. Moreover, ER stress induction impaired the activation of endothelial nitric oxide synthase (eNOS) and the angiogenic capacity of endothelial cells, while PTP1B inhibition exerted a protective effect. The results demonstrated that blockade or knockdown of PTP1B prevented ER stress‑induced apoptosis and cell cycle arrest. This effect was associated with reduced expression levels of caspase‑12 and poly (ADP‑Ribose) polymerase1. PTP1B blockade also suppressed autophagy activated by TG. The current data support the critical role of PTP1B in ER stress‑mediated endothelial dysfunction, characterized by reduced angiogenic capacity, with an underlying mechanism involving reduced eNOS activation and cell survival. These findings provide evidence of the therapeutic potential of targeting PTP1B in cardiovascular ischemic conditions.

MACC1 Contributes to the Development of Osteosarcoma Through Regulation of the HGF/c-Met Pathway and Microtubule Stability.

Osteosarcoma (OS) is the most prevalent human bone malignancy, and presents a global annual morbidity of approximately five cases per million. Notably, precise and efficient targeted therapy has become the most promising strategy for the treatment of OS; however, there is still an urgent need for the identification of suitable therapeutic targets. Metastasis-associated in colon cancer 1 (MACC1) was first identified in colon tumors by differential display RT-PCR, and was shown to be involved in the regulation of colon tumor growth and metastasis through the hepatocyte growth factor (HGF)/c-Met signaling pathway. Additionally, MACC1 overexpression has been reported to induce the growth of several types of cancers, including glioblastoma multiforme and gastric cancer. However, whether MACC1 also plays a role in the progression of OS remains unclear. In this study, we found that MACC1 was highly expressed in human OS tissues, as well as in U-2OS and MG-63 cells, when compared with normal tissues and osteoblasts, respectively. Our data further indicated that MACC1 expression was correlated with several clinicopathological features of OS. Through in vitro assays, we found that MACC1 depletion markedly suppressed the proliferative ability of both OS cells and endothelial cells, and inhibited the angiogenic capacity of endothelial cells. Similarly, MACC1 depletion inhibited tumor growth, metastasis, and angiogenesis in mice. Mechanistically, we found that MACC1 could bind to the MET promoter, and enhanced the proliferation of both OS cells and endothelial cells through the HGF/c-Met signaling pathway. Furthermore, we show that MACC1 also promoted angiogenesis by regulating microtubule dynamics, thereby promoting the progression of OS. Our results indicate that MACC1 may be a new and promising therapeutic target for the treatment of OS.

Abnormal macrophage polarization impedes the healing of diabetes-associated tooth sockets.

Patients with poorly controlled type 2 diabetes mellitus (T2DM) often experience delayed tooth extraction socket (TES) healing. Delayed healing is often associated with an aberrant inflammatory response orchestrated by either M1 pro-inflammatory or M2 anti-inflammatory macrophages. However, the precise mechanism for the attenuated TES healing remains unclear. Here we used diet-induced T2DM mice as a model to study TES. Compared with the control group, the T2DM group showed delayed TES healing and diminished expression of osteogenic and angiogenic genetic profiles. Meanwhile, we detected a more inflammatory profile, with more M1 macrophages and TNF-α expression and less M2 macrophages and PPARγ expression, in TES in the T2DM group when compared to control mice. In vitro co-culture models showed that M1 macrophages inhibited the osteogenic capacity of bone marrow stromal cells and the angiogenic capacity of endothelial cells while M2 macrophages showed an opposite effect. In addition, we constructed a gelatin/β-TCP scaffold with IL-4 to induce macrophage transformation towards M2 polarization. In vitro analyses of the hybrid scaffold revealed sustained release of IL-4 and a phenotype switch to M2 macrophages. Finally, we demonstrated that sustained IL-4 release significantly increased expression of osteogenic and angiogenic genetic profiles and improved TES healing in T2DM mice. Together, we report that increased M1 and decreased M2 macrophage polarization may be responsible for delayed TES healing in T2DM patients through abnormal expression of TNF-α and PPARγ. This imbalance negatively influences osteogenesis and angiogenesis, two of the most important biological factors in bone wound healing. Enhancing M2 macrophage polarization with IL-4 delivery system may represent a potential strategy for promoting the healing of TES in T2DM patients.

Keap1-Nrf2 signaling pathway in angiogenesis and vascular diseases.

The transcription factor, nuclear factor E2-related factor 2 (Nrf2), is highly sensitive to oxidative burst products, including reactive oxygen species (ROS) and reactive nitrogen species. In the cell nucleus, Nrf2 activates various antioxidant genes by binding to the antioxidant response elements. As an adapter for cullin 3/ring-box 1, kelch-like ECH-associated protein 1 (Keap1) ubiquitinates and degrades Nrf2 under physiological conditions. Conversely, with the aggravation of oxidative stress, Keap1-Nrf2 interaction could be much more easily dissociated. ROS play a key role in regulating the redox signaling pathway and affect the vasculature in a dose-dependent manner. Long-term production or high concentration of ROS are harmful to the vascular system, while moderate ROS can promote angiogenesis and tissue regeneration. Furthermore, appropriate regulation of oxidative stress mediated via the Keap1-Nrf2 pathway would be beneficial in various diseases associated with abnormal angiogenesis, including diabetes and cancers. Nrf2 deficiency has also been shown to result in significantly impaired survival, proliferation, and angiogenic capacity of endothelial cells in a hind limb ischemia model. Thus, this review will briefly summarize the underlying molecular mechanisms of Keap1-Nrf2 pathway in regulating oxidative stress and also help elucidate the critical role of Keap1-Nrf2 in angiogenesis under physiological and pathological conditions.

Exosomes derived from 5-fluorouracil-resistant colon cancer cells are enriched in GDF15 and can promote angiogenesis.

Background: Angiogenesis is important for tumor proliferation and distant metastasis. However, the role of drug-resistant tumor cells in angiogenesis remains largely unknown. Current anti-angiogenic strategies also have limitations and it would be useful to develop novel targets and treatment strategies.Methods: Differential ultracentrifugation was used to isolate conditioned medium-derived exosomes from 5-flurouracil (5-FU)-sensitive or -resistant colon cancer cells. Exosome endocytosis into human umbilical vein endothelial cells was observed via immunofluorescence. Differentially expressed proteins in the exosomes were confirmed via qRT-PCR and Western blotting. The angiogenic capacity of endothelial cells was evaluated using cell function assays and a rat model of abdominal aortic neovascularization. The underlying mechanisms were verified using qRT-PCR and Western blotting assays. Immunohistochemistry was used to evaluate in vivo angiogenesis.Results: We observed that the conditioned medium and exosomes from 5-FU-resistant colon cancer cells could promote angiogenesis. Exosomal growth/differentiation factor 15 (GDF15) was a potent inducer of this angiogenesis in vitro by inhibiting the Smad signaling pathway, thus increasing periostin (POSTN) levels. Moreover, 5-FU-resistant colon cancer cells showed high microvascular density in vivo. TGF-β1, an activator of the Smad signaling pathway, could partly eliminate those effects.Conclusions: Our study reveals the molecular regulation of angiogenesis in 5-FU-resistant colon cancer and suggests that the GDF15-POSTN axis may be a novel target for anti-angiogenic therapies in colon cancer.

Histatin‐1 counteracts the cytotoxic and antimigratory effects of zoledronic acid in endothelial and osteoblast‐like cells

Zoledronic acid, the most frequent agent associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ), has been reported as cytotoxic for bone and vascular cells. Hence, identification of novel approaches aiming to counteract its cytotoxic effects will be desirable to develop preventive therapies for BRONJ. The salivary peptide Histatin-1 was recently shown to promote oral wound healing, by acting in epithelial and endothelial cells; however, its effects on cells exposed to zoledronic acid have not been explored. This study aims to unveil the role of Histatin-1 in osteoblastic and vascular cell lineages challenged with zoledronic acid. The effects of zoledronic acid (1-100µM), Histatin-1 (10µM), or their combination was evaluated in cytotoxicity (Trypan Blue exclusion) and cell migration (Boyden Chamber) assays. Caspase-3 cleavage was evaluated by Western blot. The angiogenic capacity of endothelial cells was assessed in a tubule formation assay in vitro. Zoledronic acid decreased cell viability and migration of osteosarcoma cells (SAOS-2) and preosteoblasts (MC3T3-E1), in a dose-response manner. Importantly, Histatin-1 restored both cell viability and migration in both cell lines upon challenge with zoledronic acid. These effects were recapitulated in endothelial cells (EA.hy926), as Histatin-1 counteracted cytotoxic and antimigratory effects of zoledronic acid, and restored the angiogenic capacity in vitro. We conclude that Histatin-1 counteracts the cytotoxic and antimigratory effects of zoledronic acid in osteoblast-like and endothelial cells. These observations highlight the potential use of Histatin-1, in the design of novel therapies aiming to prevent and treat BRONJ.

Exosomes secreted by endothelial progenitor cells accelerate bone regeneration during distraction osteogenesis by stimulating angiogenesis

BackgroundDistraction osteogenesis (DO) is an effective but lengthy procedure to fully induce bone regeneration in large bone defects. Accumulating evidence supports the role of exosomes secreted by endothelial progenitor cells (EPC-Exos) in stimulating angiogenesis, which is closely coupled with osteogenesis. This study aimed to investigate whether EPC-Exos promote bone regeneration during DO in rats.MethodsExosomes were isolated from the supernatants of rat bone marrow EPCs via ultracentrifugation and characterized via transmission electron microscopy, tunable resistive pulse sensing analysis, and western blot analysis. Unilateral tibial DO models were generated using 68 Sprague-Dawley rats with a distraction rate of 0.5 mm per day for 10 days. After local injection of EPC-Exos into the distraction gaps after distraction, the therapeutic effects of EPC-Exos on bone regeneration and angiogenesis were assessed via X-ray, micro-computed tomography (micro-CT), and biomechanical and histological analyses. Pro-angiogenic effects and the potential mechanism underlying the effects of EPC-Exos on human umbilical vein endothelial cells were subsequently evaluated via in vitro assays including Cell Counting Kit-8, wound healing, tube formation, and western blot assays.ResultsEPC-Exos were spherical or cup-shaped vesicles ranging from 50 to 150 nm in diameter and expressed markers including CD9, Alix, and TSG101. X-ray, micro-CT, and histological analyses revealed that bone regeneration was markedly accelerated in rats treated with EPC-Exos. The distracted tibias from the Exos group also displayed enhanced mechanical properties. Moreover, vessel density was higher in the Exos group than in the control group. In addition, in vitro analyses revealed that EPC-Exos enhanced the proliferation, migration, and angiogenic capacity of endothelial cells in an miR-126-dependent manner. Further, EPC-Exos downregulated SPRED1 and activated Raf/ERK signaling.ConclusionsThe present results show that EPC-Exos accelerate bone regeneration during DO by stimulating angiogenesis, suggesting their use as a novel method to shorten the treatment duration of DO.

Influence of rabbit notochordal cells on symptomatic intervertebral disc degeneration: anti-angiogenic capacity on human endothelial cell proliferation under hypoxia.

Symptomatic degenerative disc disease (DDD) is associated with neovascularization and nerve ingrowth into intervertebral discs (IVDs). Notochordal cells (NCs) are key cells that may lead to regeneration of IVDs. However, their activities under conditions of hypoxia, the real environment of IVD, are not well known. We hypothesized that NCs may inhibit neovascularization by interacting with endothelial cells (ECs) under hypoxia. Human IVDs were isolated and cultured to produce nucleus pulposus (NP) cell conditioned medium (NPCM). Immortalized human microvascular ECs were cultured in NPCM with notochordal cell-rich rabbit nucleus pulposus cells (rNC) under hypoxia. Vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), and interleukin-8 (IL-8) were analyzed by ELISA. Focal adhesion kinase (FAK), filamentous actin (F-actin), and platelet-derived growth factor (PDGF) were evaluated to investigate EC activity. Wound-healing migration assays were performed to examine EC migration. The VEGF level of EC cells cultured in NPCM was significantly higher under hypoxia compared to normoxia. VEGF expression was significantly decreased, and FAK, F-actin, PDGF expression were inhibited when ECs were cocultured with rNCs under hypoxia. ECs cocultured with rNC in NPCM showed significantly decreased migratory activity compared to those without rNC under hypoxia. The angiogenic capacity of ECs was significantly inhibited by NCs under hypoxia via a VEGF-related pathway. Our results suggest that NCs may play a key role in the development of IVDs by inhibiting vascular growth within the disc, and this may be a promising novel therapeutic strategy for targeting vascular ingrowth in symptomatic DDD.

TIEG1 deficiency confers enhanced myocardial protection in the infarcted heart by mediating the Pten/Akt signalling pathway.

The transforming growth factor (TGF)-β-inducible early gene-1 (TIEG1) plays a crucial role in modulating cell apoptosis and proliferation in a number of diseases, including pancreatic cancer, leukaemia and osteoporosis. However, the functional role of TIEG1 in the heart has not been fully defined. In this study, we first investigated the role of TIEG1 in ischaemic heart disease. For in vitro experiments, cardiomyocytes were isolated from both TIEG1 knockout (KO) and wile-type (WT) mice, and the apoptotic ratios were evaluated after a 48-h ischaemic insult. A cell proliferation assay was performed after 7 days of incubation under normoxic conditions. In addition, the angiogenic capacity of endothelial cells was determined by tube formation assay. For in vivo experiments, a model of myocardial infarction (MI) was established using both TIEG1 KO and WT mice. Echocardiography was performed at 3 and 28 days post-MI, whereas the haemodynamics test was performed 28 days post-MI. Histological analyses of apoptosis, proliferation, angiogenesis and infarct zone assessments were performed using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) staining, BrdU immunostaining, α-smooth muscle actin (α-SMA)/CD31 immunostaining and Masson's trichrome staining, respectively. Changes in the expression of related proteins caused by TIEG1 deficiency were confirmed using both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Our results demonstrated that the absence of TIEG1 prevented cardiomyocytes from undergoing apoptosis and promoted higher proliferation; it stimulated the proliferation of endothelial cells in vitro and in vivo. Improved cardiac function and less scar formation were observed in TIEG1 KO mice, and we also observed the altered expression of phosphatase and tensin homolog (Pten), Akt and Bcl-2/Bax, as well as vascular endothelial growth factor (VEGF). On the whole, our findings indicate that the absence of TIEG1 plays a cardioprotective role in ischaemic heart disease by promoting changes in Pten/Akt signalling.

Mitochondria and Angiogenesis.

Angiogenesis is a dynamic and energy-consuming process, requiring endothelial cells to switch from a quiescent state to a migratory and proliferative phenotype in order to support the formation of new blood vessels. Despite their proximity to oxygenated blood endothelial cells are adept at utilizing glycolysis as an energy source to the detriment of mitochondrial oxidative phosphorylation. In this context, endothelial mitochondria have emerged as signaling hubs that modulate a wide range of endothelial functions, including angiogenesis, by coordinating reactive oxygen species and calcium signaling, metabolism and apoptosis. In this chapter we present an overview of the mitochondrial functions implicated in promoting or hindering the angiogenic capacity of endothelial cells, with emphasis on the mitochondrial proteins directly linked to angiogenesis. We also focus on recent findings identifying mitochondrial targeting compounds that exhibit pro-angiogenic or anti-angiogenic properties, and could therefore be of clinical importance for the treatment of vascular pathologies.

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis.

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

Endothelial Dimethylarginine Dimethylaminohydrolase 1 Is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis.

Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis and a risk factor for cardiovascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes are responsible for ADMA breakdown. It has been reported that endothelial DDAH1 accounts for the majority of ADMA metabolism. However, we and others have shown strong DDAH1 expression in a range of nonendothelial cell types, suggesting that the endothelium is not the only site of metabolism. We have developed a new endothelium-specific DDAH1 knockout mouse (DDAH1(En-/-)) to investigate the significance of endothelial ADMA in cardiovascular homeostasis. DDAH1 deletion in the DDAH1(En-/-) mouse was mediated by Tie-2 driven Cre expression. DDAH1 deletion was confirmed through immunocytochemistry, whereas Western blotting showed that DDAH1 remained in the kidney and liver, confirming expression in nonendothelial cells. Plasma ADMA was unchanged in DDAH1(En-/-) mice, and cultured aortas released amounts of ADMA to similar to controls. Consistent with these observations, vasoreactivity ex vivo and hemodynamics in vivo were unaltered in DDAH1(En-/-) mice. In contrast, we observed significantly impaired angiogenic responses both ex vivo and in vivo. We demonstrate that endothelial DDAH1 is not a critical determinant of plasma ADMA, vascular reactivity, or hemodynamic homeostasis. DDAH1 is widely expressed in a range of vascular and nonvascular cell types; therefore, the additive effect of DDAH1 expression in multiple organ systems determines plasma ADMA concentrations. Endothelial deletion of DDAH1 profoundly impairs the angiogenic capacity of endothelial cells, indicating that intracellular ADMA is a critical determinant of endothelial cell response.

Abstract 1021: JFD, a novel small molecule for inhibiting vascular endothelial growth factor receptor-mediated angiogenesis

Abstract Angiogenesis is the process of generating new capillary of blood vessels from an existing vasculature that plays a vital role in tumor growth and metastasis. Binding of vascular endothelial growth factor (VEGF) to tyrosine kinase receptor (VEGFR) is an important step for initiating vasculogenesis via. transphosphorylation of VEGF receptor associated tyrosine kinase. Therefore, blocking VEGFR or associated kinases has become a common strategy for arresting tumor angiogenesis. The newly discovered JFD is a novel anti-angiogenic agent that can limit the angiogenic capacity of endothelial cells and block tumor angiogenesis. The In vitro anti-angiogenesis potential of JFD was examined by ECMatrigelTM assay with Human umbilical vein endothelial cells (HUVEC) using different concentrations of JFD in the range of 0 - 10 µM. Among the different concentrations used, 1µM JFD significantly inhibited the angiogenesis when compared to control and a complete inhibition was observed at 10 µM concentration. Competitive binding of JFD against Fluorokine labeled VEGF for the same VEGFR was studied at 0.001 - 1.0 µM concentrations of JFD. A dose dependent inhibition of Fluorokine conjugated VEGF binding to its specific receptor in HUVEC cells was confirmed in our study. In the competition binding assay 1.0 µM concentration of JFD caused 93.5% inhibition of conjugated VEGF binding. The same 1.0 µM concentration of JFD was able to inhibit VEGF (10 ng/ml) induced phosphorylation of VEGFR2 in HUVEC cells. Furthermore, the potency of JFD as inhibitor of angiogenesis was experimentally proved by treating athymic nude mice that was implanted with GI-101A (human breast adenocarcinoma) xenograft tumor. Intraperitoneal injection of JFD at 12 mg/kg of body weight was able to significantly inhibit the tumor growth compared to the control group at different time periods. At the end of the treatment period 70% inhibition of tumor growth was observed in JFD treated animals along with prolongation of the survival. The plasma concentrations of JFD was also measured in Balb-C mice at different time periods (0 min - 180 min), using HPLC method. The data from both in vitro and in vivo study provide a strong basis for JFD to be used as a potent anti-angiogenic agent. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Sivanesan Dhandayuthapani, Appu Rathinavelu. JFD, a novel small molecule for inhibiting vascular endothelial growth factor receptor-mediated angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1021. doi:10.1158/1538-7445.AM2014-1021