Capillary-like Tubular Structures Research Articles

Tumor-expressed B7-H3 promotes vasculogenic mimicry formation rather than angiogenesis in non-small cell lung cancer.

Vasculogenic mimicry (VM), an alternative microvascular circulation independent ofangiogenesis, is formed by aggressive cancer cells. Tumor-expressedB7-H3 has been reported to promote VM formation in hepatocellular carcinoma and modulate angiogenesis in breast cancer and colorectal cancer. However, its effects on VM generation and angiogenesis in non-small cell Lung cancer (NSCLC) remained to be elucidated. CRISPR/Cas9-mediated B7-H3 knockout (KO)was conducted in NSCLC A549 and H3255 cells. The expression of VM-related proteins, including vascular endothelial (VE)-cadherin and matrix metalloproteinase 14 (MMP14), and the secretion of vascular endothelial growth factor (VEGF) were measured by western blotting and chemiluminescence assay in both B7-H3 KO and mock-edited A549 and H3255 cells. To examine VM formation, a three-dimensional (3D)culture model was used for B7-H3 KO and mock A549 and H3255 cells. For in vivo analysis, xenograft mice models were established using B7-H3 KO and mock-edited A549 cells, and immunohistochemical (CD31) and histochemical (periodic acid-Schiff, PAS) double staining were performed to identify VM and endothelial vessels in tumor tissues. Finally, specific signaling inhibitors were used to analyze B7-H3-induced signaling pathway responsible for VE-cadherin and MMP14 expression and VM generation. Higher expression of B7-H3 was associated with a worse prognosis and moreadvanced T-category in NSCLC. CRISPR/Cas9-mediated B7-H3 KOin A549 and H3255 cells led to decreased expression of VE-cadherin and MMP14; however,the secretion of VEGFby the two cell lines remained unchanged. In the3D cell culture model, bothB7-H3 KO A549 and H3255cells showed a significant reduction in the formation of capillary-like tubular structures compared to mock-edited cells. In thein vivo xenograft model, mock-edited A549 cells formed excessive PAS+ CD31-VM channels, while B7-H3 KO restrained VM formation in the xenograft tumors. However, no significant differenceswere found in CD31+ endothelial vessels between xenografts formed by B7-H3 KO and mock-edited A549 cells. Finally, we analyzed the signaling pathway responsible for B7-H3-induced VM formation and found that selective inhibition of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) hyperactivation by LY294002 was associated with decreased expression of MMP14 and VE-cadherin, and in vitro VM formation by both A549 and H3255 cells. Tumor-expressed B7-H3 acts via PI3K/AKT signaling pathway to promote VM formation by NSCLC cells while bears no effects on angiogenesis in NSCLC.

MiR-199b-5p Suppresses Tumor Angiogenesis Mediated by Vascular Endothelial Cells in Breast Cancer by Targeting ALK1.

Angiogenesis is a crucial event during cancer progression that regulates tumor growth and metastasis. Activin receptor-like kinase 1 (ALK1), predominantly expressed in endothelial cells, plays a key role in the organization of neo-angiogenic vessels. Therapeutic targeting of ALK1 has been proposed as a promising strategy for cancer treatment, and microRNAs (miRNAs) are increasingly being explored as modulators of angiogenesis. However, the regulation of ALK1 by miRNAs is unclear. In this study, we identified that ALK1 is directly targeted by miR-199b-5p, which was able to inhibit angiogenesis in vitro and in vivo. Moreover, it was found that miR-199b-5p was repressed in breast cancer cells and its expression was decreased during the VEGF-induced angiogenesis process of human umbilical vein endothelial cells (HUVECs). Overexpression of miR-199b-5p inhibited the formation of capillary-like tubular structures and migration of HUVECs. Furthermore, overexpression of miR-199b-5p inhibited the mRNA and protein expression of ALK1 in HUVECs by directly binding to its 3’UTR. Additionally, overexpression of miR-199b-5p attenuated the induction of ALK1/Smad/Id1 pathway by BMP9 in HUVECs. Finally, overexpression of miR-199b-5p reduced tumor growth and angiogenesis in in vivo. Taken together, these findings demonstrate the anti-angiogenic role of miR-199b-5p, which directly targets ALK1, suggesting that miR-199b-5p might be a potential anti-angiogenic target for cancer therapy.

442 Laminin alpha 5 in skin microvascular endothelial cell function and angiogenesis

Endothelial cell sprouting, including cell attachment and migration, and capillary vessel formation, are critical processes for angiogenesis. These activities are mediated and regulated by extracellular matrix components. Laminin α4 and α5 are two major laminin α chains expressed in human dermal microvascular endothelial cells (HDMECs). In this study, functions of laminin α5 in HDMEC adhesion, migration, capillary tubule formation in angiogenesis were investigated. Using a lentiviral pL1 vector carrying a laminin α5-specific short hairpin RNA (shRNA), laminin α5 expression in stably transduced HDMEC/pL1 cells was suppressed 70% compared to the control cells. Consequently, cell adhesive ability of HDMEC/pL1 cells was remarkably reduced 60% within an hour; and cell mobility was found decreased 83% and 71%, respectively, at 12 and 48 hours in chamber migration assays. In consistence, laminin α5 knockdown also delayed cell migration as observed in an in vitroscratched migration model, in which HDMEC/pL1 cells migrated and covered approximately 50% less area compared to the control. Moreover, depletion of laminin α5 prevented HDMEC/pL1 cells from forming enclosed capillary-like tubular structure in late stage in a three-dimensional model in vitro. In vivo, homozygous laminin α5 knockout (Lα5-/-) skin grafts was characterized with significantly reduced growth of new blood vessels. Interestingly, a concurrent and significant decrease of integrin receptors of αv and β3 has been found correlated to the laminin α5 knockdown, which implied a possible involvement of laminin α5 in integrin αvβ3-dependent angiogenesis. Furthermore, HDMEC/pL1 cells failed FAK activation during cell migration. FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. Overall, our findings suggested an important role of laminin α5 in endothelial cell functions in angiogenesis in cell attachment, migration and capillary vessel formation.

Subcellular Interactions during Vascular Morphogenesis in 3D Cocultures between Endothelial Cells and Fibroblasts.

Background: Increasing the complexity of in vitro systems to mimic three-dimensional tissues and the cellular interactions within them will increase the reliability of data that were previously collected with in vitro systems. In vivo vascularization is based on complex and clearly defined cell–matrix and cell–cell interactions, where the extracellular matrix (ECM) seems to play a very important role. The aim of this study was to monitor and visualize the subcellular and molecular interactions between endothelial cells (ECs), fibroblasts, and their surrounding microenvironment during vascular morphogenesis in a three-dimensional coculture model. Methods: Quantitative and qualitative analyses during the generation of a coculture tissue construct consisting of endothelial cells and fibroblasts were done using transmission electron microscopy and immunohistochemistry. Results: Dynamic interactions were found in cocultures between ECs, between fibroblasts (FBs), between ECs and FBs, and between the cells and the ECM. Microvesicles were involved in intercellular information transfer. FBs took an active and physical part in the angiogenesis process. The ECM deposited by the cells triggered endothelial angiogenic activity. Capillary-like tubular structures developed and matured. Moreover, some ECM assembled into a basement membrane (BM) having three different layers equivalent to those seen in vivo. Finally, the three-dimensional in vitro construct mirrored the topography of histological tissue sections. Conclusion: Our results visualize the importance of the physical contact between all cellular and acellular components of the cocultures.

MiRNA-29b suppresses tumor growth through simultaneously inhibiting angiogenesis and tumorigenesis by targeting Akt3

The traditional anti-angiogenic cancer therapy could trigger hypoxia induced factor (HIF) response, leading to “reactive resistance” to chemotherapy. Simultaneously inhibiting both angiogenesis and tumorigenesis would be ideal to overcome this limitation. MicroRNAs (miRNAs) are increasingly explored as new agents for cancer therapy. In the present study, we identified a microRNA (miR-29b) with the ability of simultaneously inhibiting angiogenesis and tumorigenesis. Ectopic expression of miR-29b inhibits HUVECs formed three-dimensional capillary-like tubular structures, tumor cell proliferation, migration and tumor formation. Systemic administration of miR-29b potently suppressed tumor vascularization and cancer cell activity in vivo, resulting in dramatic suppression of tumor growth without toxicity. Moreover, we demonstrated the role of miR-29b in anti-angiogenesis and anti-tumorigenesis is through targeting Akt3 and inducing VEGF and C-myc arrest in breast cancer cells. These findings indicate that this single miRNA could be used as an efficient anti-cancer therapeutic agent to address a critical challenge in cancer therapy.

Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation

Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me2SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.

Nanografted Substrata and Triculture of Human Pericytes, Fibroblasts, and Endothelial Cells for Studying the Effects on Angiogenesis.

For the successful treatment of severe wounds, angiogenesis must be induced to provide a sufficient blood supply to the wound site. There have been many studies on various structural and biochemical factors regulating angiogenesis, such as surface topography, surface modifications, angiogenic factors, and various cell types. However, there is a paucity of research on the effects of micro- and nanoscale topography and the application of pericytes in angiogenesis. In this study, we utilized nanoscale topography combined with a triculture system consisting of human pericytes, fibroblasts, and endothelial cells. We investigated the effects of the nanografted substrata and a triculture system on proangiogenic therapies in vitro. Human dermal fibroblasts and human umbilical vein endothelial cells were seeded with human pericytes from the placenta directly onto nanografted substrata composed of polydimethylsiloxane. We demonstrated that key elements of angiogenesis, including segment and capillary-like tubular structure formation, as well as the secretion of extracellular matrix, were increased by interaction between the nanografted substrata and the coculture containing pericytes. Thus, nanografted surfaces and triculture systems containing human pericytes, fibroblasts, and endothelial cells promote angiogenesis.

Abstract A07: Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model

Abstract Laminin-511 (composed of alpha5, beta1 and gamma1 chains) is a major extracellular matrix component in the basement membrane of human skin dermal microvascular endothelial cells (HDMECs). In this study the functions of laminin-511 in angiogenesis were investigated using in vitro knockdown and in vivo knockout skin model. In the in vitro studies, normal primary HDMECs and RNA interfering technique (siRNA) to silence the gene expression strategy were used. A lentiviral-based delivery system was developed that expresses laminin alpha5 chain specific siRNA (pL1) or control LUC siRNA (pLUC). HDMECs transduced with Laminin alpha5 siRNA (HDMEC/pL1) or control (HDMEC/pLUC) were analyzed in cell attachment, migration and a tubule formation assays. The mRNA expressions of laminin alpha5 and integrins were examined by real-time RT-PCR. Focal adhesion kinase (FAK) activation was studied by Western blot. In the in vivo study, a knockout mouse skin angiogenesis model was used. Since laminin alpha5 homozygous knockout (-/-) is embryonic lethal in mouse, skin from laminin alpha5 (-/-) embryos was transplanted onto the back of nude mouse, excisional wounds were created and wound angiogenesis was analyzed. Our results demonstrated that knockdown laminin alpha5 expression significantly inhibited the ability of endothelial cells in attachment, migration and tubule formation in vitro and blood vessel growth in vivo. Cell adhesive ability of HDMEC/pL1 was reduced 60% during a 60 minute attachment assay. Cell mobility of HDMEC/pL1 was found remarkably decreased 83% and 71% in a 12-hour and 48-hour chamber migration assay respectively; and in consistence, cell migration rates in a scratch migration assay were 56% and 53% reduced in HDMEC/pL1 cells at 48 and 72 hours respectively. Moreover, depletion of laminin alpha5 prevented HDMEC from forming enclosed capillary-like tubular structure at later stage in a three-dimensional tubule formation assay. While, in vivo, homozygous laminin alpha5 knockout skin was associated with significantly reduced growth of new blood vessels. A simultaneous and significant down regulation of integrins alphaV and beta3 was found correlated with the laminin alpha5 knockdown and reduced rate of cell migration. Further, cells with laminin alpha5 knockdown failed FAK activation, FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. In conclusion, our data suggested a possible involvement of laminin-511 in integrin alphaV and beta3-dependent angiogenesis and blood vessel maturation. Our works revealed the important roles of laminin-511 in endothelial cell activities, which proved its significance for angiogenesis. Citation Format: Jie Li, Tengjiao Cui. Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A07.

Abstract 12780: Collagen Derived Matricryptin Robustly Promotes Angiogenesis Post-myocardial Infarction Reducing Cardiac Dysfunction

Proteolytically released extracellular matrix (ECM) fragments - matricryptins - exert biological activities usually different from those of the full-length protein and play important roles in tissue healing and regeneration. Following myocardial infarction (MI), the native ECM is cleaved by proteases during remodeling of the left ventricle (LV) originating ECM-derived peptides. Previously, we identified a collagen matricryptin, p1158/59, that is naturally generated post-MI. This study aimed to identify the biological roles of this matricryptin. We isolated cardiac fibroblasts from mouse hearts and treated cells with 1 μM of p1158/59. HUVEC cells were used to assess p1158/59 effects in angiogenesis. A permanent occlusion model of MI was used to investigate the roles of p1158/59 in post-MI remodeling. Animals were treated with p1158/59 (950 μg/day/kg body weight) or saline for 7 days post-MI. In vitro, p1158/59 increased migration of cardiac fibroblasts and robustly induced formation of a network of 3-D capillary-like tubular structures in endothelial cells (~4-fold versus negative control, p<0.05). In vivo, mice treated with p1158/59 for 7 days post-MI showed enhanced expression of components of the basal membrane known to stimulate angiogenesis - Col4a1, Col4a2, and Lama1 (p<0.05 for all). By immunoblotting and immunohistochemistry we observed increased vessel numbers in both the remote and infarct regions of p1158/59 treated animals compared to those treated with saline (p<0.05). Increased angiogenesis in both remote and infarct regions may offer the necessary cardioprotection to mitigate the detrimental effects of hypoxia and resulting LV dysfunction post-MI. We used echocardiography to assess physiological effects. Animals treated with p1158/59 displayed preserved LV wall thickness, geometry, and contractility, resulting in reduced systolic dysfunction post-MI. Taken together, our data show that p1158/59 is a biologically active molecule with potent pro-angiogenic properties and strongly promote this matricryptin as a molecule of interest for treatment of adverse LV remodeling.

MARCKSL1 exhibits anti-angiogenic effects through suppression of VEGFR-2-dependent Akt/PDK-1/mTOR phosphorylation.

Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) plays a pivotal role in the regulation of apoptosis and has been shown to maintain antitumor and metastasis-suppressive properties. In the present study, we examined the effects of MARCKSL1 as a novel anti-angiogenic agent on the inhibition of angiogenesis-mediated cell migration. MARCKSL1 also reduced vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, as well as capillary-like tubular structure formation in vitro. MARCKSL1 disrupted phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) in ovarian tumorigenesis. In addition, MARCKSL1 showed potent anti-angiogenic activity and reduced the levels of VEGF and hypoxia-inducible factor 1α (HIF-1α) expression, an essential regulator of angiogenesis. Consistently, MARCKSL1 decreased VEGF‑induced phosphorylation of the PI3K/Akt signaling pathway components, including phosphoinositide-dependent protein kinase 1 (PDK-1), mammalian target of rapamycin (mTOR), tuberous sclerosis complex 2 (TSC-2), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3β (GSK-3β) protein. Collectively, our results provide evidence for the physiological/biological function of an endothelial cell system involved in angiogenesis through suppression of Akt/PDK-1/mTOR phosphorylation by interaction with VEGFR-2.

SMEK1 inhibits endothelial cell proliferation by attenuating VEGFR-2-dependent-Akt/eNOS/HIF-1α signaling pathways.

The suppressor of MEK null (sMEK1) protein possesses pro-apoptotic activities. In the current study, we reveal that sMEK1 functions as a novel anti-angiogenic factor by suppressing vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, and capillary-like tubular structure in vitro. In addition, sMEK1 inhibited the phosphorylation of the signaling components up- and downstream of Akt, including phospholipase Cγ1 (PLC-γ1), 3-phosphoinositide-dependent protein kinase 1 (PDK1), endothelial nitric oxide synthetase (eNOS), and hypoxia-inducible factor 1 (HIF-1α) during ovarian tumor progression via binding with vascular endothelial growth factor receptor 2 (VEGFR-2). Furthermore, sMEK1 decreased tumor vascularity and inhibited tumor growth in a xenograft human ovarian tumor model. These results supply convincing evidence that sMEK1 controls endothelial cell function and subsequent angiogenesis by suppressing VEGFR-2-mediated PI3K/Akt/eNOS signaling pathway. Taken together, our results clearly suggest that sMEK1 might be a novel anti-angiogenic and anti-tumor agent for use in ovarian tumor.

Coimplanted Endothelial Cells Improve Adipose Tissue Grafts’ Survival by Increasing Vascularization

With goal of improving fat graft survival, many studies have focused on supplementing cells in the graft fat. In these studies, enhanced vascularization is considered the most important mechanism for the improved graft survival. Endothelial cells (ECs) are essential in vessel formation of the vascularization. Therefore, in this study, we coimplanted ECs with adipose tissue to investigate whether the ECs can enhance graft survival in a cell concentration-dependent manner. Endothelial cells were isolated from stromal vascular fraction derived from human liposuction aspirates, and the EC characteristics were confirmed by CD31 immunofluorescence staining, measuring acetylated low-density lipoprotein uptake, and observing the formation of capillary-like tubular structures in Matrigel. During the animal experiment, the isolated ECs were labeled, then added to 0.5-mL fat grafts at different numbers (0.5 × 10(6), 1 × 10(6), 2 × 10(6), and 4 × 10(6) cells) before subcutaneous implantation in nude mice. Grafts were harvested at 1 week, 1 month, and 2 months after -transplantation, and graft survival and vascularization were evaluated based on weight measurements, histological assessment, and vascular gene expression. Stromal vascular fraction-derived vascular cells exhibited typical EC characteristics. The observed differences in explanted graft weight, vessel density, vascular gene expression, and cell tracking result indicated that coimplantation with ECs accelerated vascularization that increased graft survival in a concentration-dependent manner. Over the experimental period, fat grafts implanted with 4 × 10(6) ECs showed no weight loss and the greatest increases in measures of vascularization. Endothelial cells can effectively enhance vascularization in fat grafts, and higher EC concentrations (eg, 4 × 10(6) ECs/0.5 mL adipose tissue) may best support graft survival.

Abstract 1737: Allosteric FGFR2 inhibitor RPT835 impacts on tumor growth and neovascularization

Abstract Encouraged by the clinical validation of fibroblast growth factor receptor 2 (FGFR2) as the target for the treatment of several tumors, we sought to identify pharmacological agents that could target this receptor by an allosteric mechanism. We report the efficacy of RPT835, novel highly potent inhibitor of the FGFR2 extracellular domain (RusPharmTech, LLC), in vitro and in vivo. Concordant with this activity, in human umbilical vein endothelial cells (HUVEC) and mouse endothelial cells (SVEC-4-10), RPT835 inhibited FGF-stimulated proliferation with IC50 values of 11 and 10 nmol/L, respectively. Inhibition of HUVEC cells proliferation by brivanib as positive control was significantly lower (IC50=289 nmol/L, P<0.001). Once-daily oral administration of RPT835 significantly ablated experimental FGF-induced angiogenesis in vivo and reduced endothelial cell migration, capillary-like tubular structure and mature vessel formation in comparison with bevacizumab (P<0.001). The growth of established human tumor xenograft of breast cancer (HS578T) in athymic mice was inhibited by RPT835 with chronic administration of 30 mg per kg per day. Differences with control group were significant on 34 day of study. There was no significant impact of RPT835 on colon (HCT-116, 10 mg/kg) and lung (NCI-H226, 30 mg/kg) cancer growth. Collectively, the data obtained with RPT835 are consistent with potent inhibition of FGF signaling, angiogenesis, and breast cancer growth. Citation Format: Ilya Tsimafeyeu, Evgenia Stepanova, Eliso Solomko, Dmitry Kochenkov, Nina Peretolchina, Oxana Ryabaya, Mikhail Byakhov, Sergey Tjulandin. Allosteric FGFR2 inhibitor RPT835 impacts on tumor growth and neovascularization. [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 1737. doi:10.1158/1538-7445.AM2014-1737

Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.

OM-RCA-01, a novel humanized monoclonal antibody targeting fibroblast growth factor receptor 1, in renal cell carcinoma model

Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic renal cell carcinoma (RCC). We investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody in RCC. OM-RCA-01 has been shown to inhibit in vitro kinase activity of FGFR1 and has high affinity (Kd of 1.59 nM). In human renal carcinoma Caki-1 FGFR1-expressing cells, OM-RCA-01 potently inhibited FGF-mediated signaling and proliferation. In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2,000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (P < 0.01) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (P = 0.917). In Matrigel assay, OM-RCA-01 significantly inhibited FGF-induced endothelial cell migration, capillary-like tubular structure and mature vessels formation. Administration of 10 mg/kg antibody for up to 35 days resulted in minimal body weight loss and no observations of gross toxicity were made. Collectively, the data obtained with OM-RCA-01 are consistent with potent inhibition of FGFR1-signaling, angiogenesis, and tumor growth. OM-RCA-01 is being developed clinically as an intravenous therapy for the treatment of clear cell RCC.

Isolation and characterization of endothelial cells from intramuscular hemangioma

BackgroundIntramuscular hemangiomas (IMHs) are benign vascular tumors of deep soft tissue characterized by endothelial cell (EC) proliferation. The purpose of this study was to isolate ECs from IMH, characterize their angiogenic phenotype and functional characteristics, and search for a possible signaling pathway related to IMH development. MethodsEC Isolation from IMH was performed by digestion, filtration, washing, incubation, and purification in sequence. Tie2 expression was compared between ECs from IMH and controls using reverse transcriptase polymerase chain reaction (RT-PCR). Cell invasion and proliferation assays were used to analyze functional responses of ECs to angiopoietin 1 (Ang1) and vascular endothelial growth factor (VEGF). Expression of downstream targets was analyzed using Western blot analysis. ResultsIsolated ECs showed typical cobblestone appearance under light microscopy and formed capillary-like tubular structures using Matrigel tube-forming assay. RT-PCR of isolated ECs from six patients showed increased expression of Tie2 and VEGF receptor 1 (VEGFR1) compared with control ECs. Tie2 activation by Ang1 compared with VEGFR1 by VEGF resulted in increased EC migration and proliferation. Western blot analysis showed increased Tie2 expression in hemangioma samples compared with normal ECs. Phosphorylated Akt and phosphorylated forkhead box O1 (FOXO1) expression was observed in hemangioma samples only. ConclusionEC isolation from IMH could be a useful tool for further research. These results suggest that increased Tie2 expression, via Akt-FOXO1 pathway activation, may play an important role in IMH pathogenesis.

In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

[Abstract] Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to their receptors, release of proteases to dissolve the basement membrane, migration towards an angiogenic signal, proliferation, and an increase in cell number for new blood vessel formation. Finally, reorganization of ECs forms the three-dimensional vasculature. HUVEC tube-formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of ECs to form three-dimensional capillary-like tubular structures, when cultured on a gel of growth factor-reduced basement membrane extracts. During the assay, ECs differentiate, directionally migrate to align, branch, and form the tubular polygonal networks of blood vessels.

Adipose-derived stem cells (ASCs) as a source of endothelial cells in the reconstruction of endothelialized skin equivalents

Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models.

Propranolol suppresses angiogenesis in vitro: Inhibition of proliferation, migration, and differentiation of endothelial cells

Propranolol, a non-selective β-adrenergic blocking drug, was recently reported to control the growth of hemangiomas, the most common vascular tumor of infancy. However, the mechanisms involved in this effect remain unknown. Here, we demonstrate that propranolol dose-dependently inhibited growth factor-induced proliferation of cultured human umbilical vein endothelial cells (HUVECs) through a G 0/G 1 phase cell cycle arrest. This was correlated to decreased cyclin D1, cyclin D3, and cyclin-dependent kinase CDK6 protein levels, while increases in the CDK inhibitors p15 INK4B, p21 WAF1/Cip1 and p27 Kip1 were observed. Chemotactic motility and differentiation of HUVECs into capillary-like tubular structures in Matrigel were also inhibited by propranolol. Furthermore, inhibition by propranolol of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor-2 lead to inhibition of downstream signaling such as the activation of the extracellular signal-regulated kinase-1/2 and the secretion of the extracellular matrix degrading enzyme MMP-2. Taken together, these results demonstrate that propranolol interferes with several essential steps of neovascularization and opens up novel therapeutic opportunities for the use of β-blockers in the treatment of angiogenesis-dependent human diseases.

Morphologic features of biocompatibility and neoangiogenesis onto a biodegradable tracheal prosthesis in an animal model

We evaluated a newly designed bioresorbable polymer (Degrapol) tracheal prosthesis in an in-vivo angiogenesis-inducing animal model focusing on the specific tissue reaction, the neo-angiogenesis and also the eventual cathepsin B role during the polymer degradation. Fifteen rabbits were divided into three groups (2, 6 and 8 weeks) and our tube-shaped porous prosthesis was implanted using the common carotid artery and the internal jugular vein as vascular pedicle. Optical and electron microscopy, immunohistochemistry and immunocytochemistry were performed at the end of each period, showing cells and fibrils, in direct contact with the Degrapol scaffold, strongly increased with time. Blood vessel neoformation was visible with CD31 expression localized at the endothelial cells forming the neovascular walls. Over time many of them differentiate in muscle fibers as validated by the expression of alpha-smooth muscle actin (SMA). Few inflammatory cells, expressing CD14, were visible while most cells adopting a pronounced spreading phenotype showed a strong positivity for cathepsin B. We concluded that this bioresorbable polymer provided a good substrate for fibrous tissue deposition with an excellent degree of neo-angiogenesis. Also, cathepsin B seems to contribute to the polymer degradation and particularly to neovascularization by stimulating capillary-like tubular structures and cell proliferation.