Higher Vascular Density Research Articles

Micro-fabricated scaffolds lead to efficient remission of diabetes in mice.

Despite the clinical success of intrahepatic islet transplantation in treating type 1 diabetes, factors specific to this transplantation site hinder long-term insulin independence. The adoption of alternative, extravascular sites likely improve islet survival and function, but few locations are able to sufficiently confine islets in order to facilitate engraftment. This work describes a porous microwell scaffold with a well-defined pore size and spacing designed to guarantee islet retention at an extrahepatic transplantation site and facilitate islet revascularization. Three techniques to introduce pores were characterized: particulate leaching; solvent casting on pillared wafers; and laser drilling. Our criteria of a maximum pore diameter of 40μm were best achieved via laser drilling. Transplantation studies in the epididymal fat of diabetic mice elucidated the potential of this porous scaffold platform to restore blood glucose levels and facilitate islet engraftment. Six out of eight mice reverted to stable normoglycemia with a mean time to remission of 6.2±3.2 days, which was comparable to that of the gold standard of renal subcapsular islet grafts. In contrast, when islets were transplanted in the epididymal fat pad without a microwell scaffold, only two out of seven mice reverted to stable normoglycemia. Detailed histological evaluation four weeks after transplantation found a comparable vascular density in scaffold-seeded islets, renal subcapsular islets and native pancreatic islets. However, the vascularization pattern in scaffold-seeded islets was more inhomogeneous compared to native pancreatic islets with a higher vascular density in the outer shell of the islets compared to the inner core. We also observed a corresponding decrease in the beta-cell density in the islet core. Despite this, our data indicated that islets transplanted in the microwell scaffold platform were able to maintain a viable beta-cell population and restore glycemic control. Furthermore, we demonstrated that the microwell scaffold platform facilitated detailed analysis at a subcellular level to correlate design parameters with functional physiological observations.

EPR effect based nanocarriers targeting for treatment of cancer

<p>The enhanced permeability and retention (EPR) effect is a unique phenomenon of solid tumors related to their anatomical and pathophysiological differences from normal tissues. In solid tumors, angiogenesis leads to high vascular density. Large gaps exist between endothelial cells in tumor blood vessels, which lead to selective extravasations and retention of macromolecular drugs. This EPR effect served as a basis for development of macromolecular anticancer therapy. There are various factors, which lead to a significantly increased EPR effect and enhanced antitumor drug effects as well. This review discusses the unique anatomy of tumor vessels, molecular mechanisms of factors related to the EPR effect and the role of the EPR effect in the intratumoral delivery of protein and peptide drugs, macromolecular drugs and drug-loaded long-circulating nanocarriers.</p>

Tumor angiogenesis of SCLC inhibited by decreased expression of FMOD via downregulating angiogenic factors of endothelial cells.

Fibromodulin (FMOD), an ECM small leucine-rich proteoglycan (SLRP), was reported to promote angiogenesis not only during wound healing, but also in optical and cutaneous angiogenesis-dependent diseases. However, whether it plays important roles in tumor angiogenesis remains unclear. To explore the role of FMOD in tumor angiogenesis of human small cell lung cancer (SCLC), initially the study analyzed the relationship of FMOD expression in cancer tissues of SCLC with clinical characteristics. The analysis revealed that the positive FMOD expression was significantly associated with extensive stage of SCLC and higher vascular density. In mouse models, xenograft tumors developed with FMOD-silenced H446 cells (H446-shFMOD) exhibited slowed growth rate, decreased microvessel density, and reduced blood perfusion related to that of controls (H446-shCON). Additionally, compared with that of controls, the decreased secretion of FMOD in conditioned medium (CM) from H446-shFMOD inhibited proliferation, migration, and invasion of human umbilical vessel endothelial cells (HUVECs). Moreover, the decreased secretion of FMOD downregulated the expression of VEGF, TGF-β1, FGF-2, and PDGF-B in HUVECs. The findings strongly suggested that the autocrine FMOD of cancer cells may promote tumor angiogenesis of SCLC by upregulating the expression of angiogenic factors that act in concert to facilitate the angiogenic phenotype of endothelial cells as a proangiogenic factor. Therefore, silencing FMOD may be a potentially clinical therapy for repressing tumor angiogenesis.

Epigallocatechin-3-gallate promotes angiogenesis via up-regulation of Nfr2 signaling pathway in a mouse model of ischemic stroke

Epigallocatechin-3-gallate (EGCG) is the major effective component of green tea and has been known as a potential anticancer drug because of its antioxidant and anti-angiogenic properties. EGCG has also been reported to have preventive effects against ischemic stroke via nuclear factor erythroid 2-related factor 2 (Nfr2) signaling pathway, but how EGCG affect angiogenesis after stroke remains unclear. In this study, we investigated whether EGCG treatment in the acute phase of ischemic stroke can promote angiogenesis in a mouse model of transient middle cerebral artery occlusion (MCAO). We assessed neurological function with modified neurologic severity score (mNSS) test, infarct volume by Nessl staining, angiogenesis and oxidative stress by immunofluorescence analysis, intravital lectin perfusion analysis, western blot analysis and enzyme-linked immunosorbent assay (ELISA). In order to explore the role of Nrf2 in the angiogenesis of MCAO+EGCG-treated mice, we used MAPK/ERK inhibitor PD98059 to block the activation of Nrf2. We found MCAO+EGCG-treated mice had better neurologic outcome, less infarct volume, more number of Ki67/CD31-positive vessels, higher vascular density, unregulated VEGF-VEGFR2 signaling pathway, increased Nrf2 expression and decreased oxidative stress than did MCAO+vehicle-treated mice. Blocking Nrf2 with PD98059 significantly reduced the expression of Nrf2, increased oxidative stress and abolished the angiogenic and neuroprotective effects of EGCG on MCAO mice. We conclude that EGCG treatment in the early stage of ischemic stroke can promote angiogenesis in MCAO mice, possibly via upregulation of Nrf2 signaling pathway.

In-vivo quantification of the revascularization of a human acellular dermis seeded with EPCs and MSCs in co-culture with fibroblasts and pericytes in the dorsal chamber model in pre-irradiated tissue.

Transplantation of a cell-seeded graft may improve wound healing after radiotherapy. However, the survival of the seeded cells depends on a rapid vascularization of the graft. Co-culturing of adult stem cells may be a promising strategy to accelerate the vessel formation inside the graft. Thus, we compared the in vivo angiogenic potency of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) using dorsal skinfold chambers and intravital microscopy. Cells were isolated from rat bone marrow and adipose tissue and characterized by immunostaining and flow cytometry. Forty-eight rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and non-irradiated. Each of these 2 groups were further subdivided into 4 groups: unseeded matrices, matrices+fibroblasts+pericytes, matrices+fibroblasts+pericytes+MSCs and matrices+fibroblasts+pericytes+EPCs. Vessel densities were quantified semi-automatically using FIJI. Fibroblasts+pericytes-seeded matrices showed a significantly higher vascular density in all groups with an exception of non-irradiated rats at day 12 compared to unseeded matrices. Co-seeding of MSCs increased vessel densities in both, irradiated and non-irradiated groups. Co-seeding with EPCs did not result in an increase of vascularization in none of the groups. We demonstrated that the pre-radiation treatment led to a significant decreased vascularization of the implanted grafts. The augmentation of the matrices with fibroblasts and pericytes in co-culture increased the vascularization compared to the non-seeded matrices. A further significant enhancement of vessel ingrowth into the matrices could be achieved by the co-seeding with MSCs in both, irradiated and non-irradiated groups.

Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis

BackgroundDisrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. MethodsThe expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. ResultsOverexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P < .0001, r = 0.53), cathepsin D (P = .0002, r = 0.36), light chain 3A (LC3A) (P = .02, r = 0.22), and hypoxia-inducible factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. ConclusionIntense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies.

Erythropoietin improves hypoxic-ischemic encephalopathy in neonatal rats after short-term anoxia by enhancing angiogenesis

Erythropoietin (EPO) is important for angiogenesis after hypoxia/ischemia. In this study, we investigated whether recombinant human erythropoietin (rhEPO) can enhance angiogenesis, and promote cognitive function through vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of hypoxic-ischemic encephalopathy (HIE). RhEPO, selective VEGFR2 inhibitor (SU5416) or vehicle was administrated by intraperitoneal injection. The assessment for cognitive function begins on day 60 after anoxia. Vascular density in hippocampus and white matter damage within corpus callosum were examined on day 28 after anoxia. The expression of erythropoietin receptor (EPOR), VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in hippocampus were evaluated on day 7 after anoxia. RhEPO-treated anoxia rats had better cognitive recovery, higher vascular density, and less white matter damage than in the vehicle anoxia rats. These protective effects associated with increased expression of EPOR, VEGF; and increased phosphorylation of Raf1 and ERK1/2. While this up-regulation, and changes in the histopathologic and functional outcomes were abolished by SU5416. Our data indicate that rhEPO can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery through VEGF/VEGFR2 signaling pathway in anoxia rats.

Hypoxia induced CCL28 promotes angiogenesis in lung adenocarcinoma by targeting CCR3 on endothelial cells.

Tumor hypoxia is one of the important features of lung adenocarcinoma. Chemokines might mediate the effects caused by tumor hypoxia. As confirmed in tumor tissue and serum of patients, CC chemokine 28 (CCL28) was the only hypoxia induced chemokine in lung adenocarcinoma cells. CCL28 could promote tube formation, migration and proliferation of endothelial cells. In addition, angiogenesis was promoted by CCL28 in the chick chorioallantoic membrane and matrigel implanted in dorsal back of athymic nude mice (CByJ.Cg-Foxn1nu/J). Tumors formed by lung adenocarcinoma cells with high expression of CCL28 grew faster and had a higher vascular density, whereas tumor formation rate of lung adenocarcinoma cells with CCL28 expression knockdown was quite low and had a lower vascular density. CCR3, receptor of CCL28, was highly expressed in vascular endothelial cells in lung adenocarcinoma when examining by immunohistochemistry. Further signaling pathways in endothelial cells, modulated by CCL28, were analyzed by Phosphorylation Antibody Array. CCL28/CCR3 signaling pathway could bypass that of VEGF/VEGFR on the levels of PI3K-Akt, p38 MAPK and PLC gamma. The effects could be neutralized by antibody against CCR3. In conclusion, CCL28, as a chemokine induced by tumor hypoxia, could promote angiogenesis in lung adenocarcinoma through targeting CCR3 on microvascular endothelial cells.

Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy

ABSTRACTPurpose: To determine the presence of Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR).Methods: Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells.Results: In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (α-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells.Conclusions: The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR.

C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa−/−) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa−/− mice. Bone marrow (BM) chimeras between C1qa−/− and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.

IGFBP7 functions as a potential lymphangiogenesis inducer in non-small cell lung carcinoma.

Lymphangiogenesis is not only involved in the processes of embryonic development, tissue repair and chronic inflammation, but also in tumor lymphatic metastasis. Metastatic tumor cells spreading through lymphatic vessels occur in non-small cell lung carcinoma (NSCLC), with regional lymph node metastasis often being the most important prognostic factor for carcinoma patients. Recent research has identified a range of lymphangiogenic growth factors that could conceivably play a great role in promoting tumor lymphangiogenesis and lymphatic metastasis. The most extensively accepted signaling pathways promoting lymphangiogenesis in tumors include the secreted lymphangiogenic proteins: vascular endothelial growth factor-C (VEGF-C) and VEGF-D, and their cognate receptor on lymphatic endothelium VEGF receptor-3 (VEGFR-3). Targeting VEGF pathway strategy sometimes failed to decrease tumor metastasis in vivo experiments and clinical trials. It is unclear whether the tumor cells induced the lymphangiogenesis process, while VEGF pathway could not completely illustrate the mechanism of tumor cell lymphatic metastasis. To explore the novel tumor lymphangiogenesis targets, we screened 181 candidate genes between high lymphatic vascular density (LVD) and low LVD in lung adenocarcinomas using Human Genome U133 Plus 2.0 Microarray. Insulin-like growth factor binding protein 7 (IGFBP7) was proven to be associated with metastatic clinicopathological features and high LVD. Furthermore, by assessing the capability of lymphatic endothelial cell forming lymphatic vessel-like structures in vitro, it appears to enhance lymphangiogenesis.

Effects of Mesenchymal Stem Cell Treatment on the Expression of Matrix Metalloproteinases and Angiogenesis during Ischemic Stroke Recovery.

BackgroundThe efficacy of mesenchymal stem cell (MSC) transplantation in ischemic stroke might depend on the timing of administration. We investigated the optimal time point of MSC transplantation. After MSC treatment, we also investigated the expression of matrix metalloproteinases (MMPs), which play a role in vascular and tissue remodeling.MethodsHuman bone marrow-derived MSCs (2 × 106, passage 5) were administrated intravenously after permanent middle cerebral artery occlusion (MCAO) was induced in male Sprague-Dawley rats. First, we determined the time point of MSC transplantation that led to maximal neurological recovery at 1 h, 1 day, and 3 days after MCAO. Next, we measured activity of MMP-2 and MMP-9, neurological recovery, infarction volume, and vascular density after transplanting MSCs at the time that led to maximal neurological recovery.ResultsAmong the MSC-transplanted rats, those of the MSC 1-hour group showed maximal recovery in the rotarod test (P = 0.023) and the Longa score (P = 0.018). MMP-2 activity at 1 day after MCAO in the MSC 1-hour group was significantly higher than that in the control group (P = 0.002), but MMP-9 activity was not distinct. The MSC 1-hour group also showed smaller infarction volume and higher vascular density than did the control group.ConclusionsIn a permanent model of rodent MCAO, very early transplantation of human MSCs (1 h after MCAO) produced greater neurological recovery and decreased infraction volume. The elevation of MMP-2 activity and the increase in vascular density after MSC treatment suggest that MSCs might help promote angiogenesis and lead to neurological improvement during the recovery phase after ischemic stroke.

All ecosystems look messy, but some more so than others: A case-study on the management and acceptance of Mediterranean urban grasslands

Abstract The spontaneous revegetation of a disused demolition waste landfill in Rome (Italy) led to a very high vascular plant species density (269 species in 0.2 km2). Vegetation was dominated by Mediterranean annual species (Therophytes), and floristic assemblage was consistent with the regional species pool. No significant levels of biotic homogenization were found. Local activists successfully campaigned to save the site from development, which resulted in its designation as an Urban Nature Reserve; however, it was essentially managed as a conventional neighbourhood park. As a consequence, vascular plant species richness decreased by 50%. The functional and biogeographic groups most typical of Mediterranean habitats saw the largest decrease (the steno-Mediterranean element decreased by 80%), leading to biotic homogenization via changes in native species only (no increases in alien species were observed). The removal of most of the old demolition-waste mounds was probably the main cause for these observed changes. These mounds provide well-drained, oligotrophic soils and micro-habitat heterogeneity. Mediterranean grassland vegetation, especially within urban areas, is often perceived as “untidy” and undesirable by local residents and decision-makers, who tend to overlook or ignore the ecological value of spontaneous grasslands—perhaps distracted by their apparently “messy” aesthetics. Mediterranean urban wastelands that are “saved” with the scope of creating public green spaces are particularly at risk of being inappropriately managed from the ecological perspective. Thus, in dry-climate regions, ecologists should be particularly careful not to dismiss the importance of aesthetic issues and cultural acceptance in urban nature conservation. Considering the results of this case-study, we offer some generalized management guidelines that can help render urban dry grasslands acceptable for recreational use, while at the same time preserving their biodiversity and educational value.

T2*-weighted fMRI time-to-peak of oxygen challenge in ischemic stroke

T2*-weighted MRI of transient oxygen challenge (OC) showed exaggerated OC percent changes in the ischemic tissue at risk compared to normal tissue. One ambiguity is that regions with high vascular density also showed exaggerated OC percent changes. This study explored time-to-peak (TTP) of the OC percent changes to improve the utility of T2*-weighted OC MRI. Experiments were performed longitudinally at 30 min, 150 min and 24 h after transient (60-min) stroke in rats. Ischemic core, normal, and mismatch tissue were classified pixel-by-pixel based on apparent diffusion coefficient and cerebral blood flow. Major findings were: (i) Delayed OC TTP was localized to and corresponded well with the perfusion-diffusion mismatch. (ii) By contrast, the exaggerated OC percent changes were less localized, with changes not only in the at-risk tissue but also in some areas of the contralesional hemisphere with venous vessel origins. (iii) The OC time-course of the mismatch tissue was biphasic, with a faster initial increase followed by a slower increase. (iv) At-risk tissue with delayed TTP and exaggerated OC was normal after reperfusion and the at-risk tissue was mostly (83 ± 18%) rescued by reperfusion as indicated by normal 24-h T2. OC TTP offers unique information toward better characterization of at-risk tissue in ischemic stroke.

Lymphangiogenic Markers and Their Impact on Nodal Metastasis and Survival in Non-Small Cell Lung Cancer--A Structured Review with Meta-Analysis.

BackgroundIn non-small cell lung cancer (NSCLC), nodal metastasis is an adverse prognostic factor. Several mediating factors have been implied in the development of nodal metastases and investigated for predictive and prognostic properties in NSCLC. However, study results differ. In this structured review and meta-analysis we explore the published literature on commonly recognized pathways for molecular regulation of lymphatic metastasis in NSCLC.MethodsA structured PubMed search was conducted for papers reporting on the expression of known markers of lymhangiogenesis in NSCLC patients. Papers of sufficient quality, presenting survival and/or correlation data were included.ResultsHigh levels of vascular endothelial growth factor C (VEGF-C, HR 1.57 95% CI 1.34–1.84) and high lymphatic vascular density (LVD, HR 1.84 95% CI 1.18–2.87) were significant prognostic markers of poor survival and high expression of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR3) and LVD was associated with lymph node metastasis in NSCLC.ConclusionLymphangiogenic markers are prognosticators of survival and correlate with lymph node metastasis in NSCLC. Their exact role and clinical implications should be further elucidated.

Abstract 4161: Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies

Abstract Previously, we demonstrated immunohistochemical expression of VEGFR2 protein and vascular phenotypes in human breast carcinomas (Holzer, Nasir et al AACR 2014). Here, we carried out a large-scale immunopathologic analysis of tumor vascular density and differential expression of VEGFR2 protein in various subsets of primary human breast carcinomas from 186 females (Mean age: 59 years; range 33-88 years). Sections from a primary breast cancer tissue microarray (Yale University), were stained for a sensitive tumor vascular marker (CD34) and for VEGFR2 protein using a novel VEGFR2 IHC assay developed and standardized in our laboratory (Holzer, Nasir et al PLOS One 2013). Discrete VEGFR2+ and CD34+ tumor vessels were counted in each analyzable TMA core by an experienced breast pathologist, excluding any suboptimal/inadequate cores or any areas of tumor necrosis. Histologic subtypes of primary breast carcinomas included invasive ductal, lobular, mixed ductal-lobular and colloid (N = 139, 22, 18, 7) respectively. Based on hormone receptor (HR) and Her2 status, cases were grouped into HR+, Her2+ and triple-negative subsets. Pathologic assessments of vascular density (CD34 counts) and imunohistochemical expression of VEGFR2 protein were made only in the viable invasive carcinoma tissue. The observed CD34+ and VEGFR2+ tumor vascular counts in individual cases were heterogeneous, ranging from high vascular density and high VEGFR2 expression to minimal vascular density and/or minimal or no VEGFR2 expression. Among all histologies, mean CD34+ and VEGFR2+ tumor vascular counts were 11 and 3.4 per tumor TMA core respectively. Eighty-nine of 186 (48%) cases had &amp;gt;10 CD34+ tumor vessels, while 97/186 (52%) had fewer CD34+ vessels in each TMA core. Of 169 analyzable cores in the VEGFR2 stained TMA, 90 (53%) showed 1 to 5 VEGFR2+ tumor vessels/TMA core, while 42/169 (25%) cores had no VEGFR2+ tumor vessels. Thirteen of 169 (8%) cases also showed tumor cell (cytoplasmic and/or membrane) expression of VEGFR2 protein. Triple-negative breast cancers (TNBCs) appeared to be less vascular (Mean VD = 9.8, range 0-34) than other breast cancer subtypes. Overall, VEGFR2+ tumor vessel counts were significantly higher in Her2+ as compared to HR+ (p = 0.03) and TNBC (p = 0.01) tissues. Compared to Her2- cases, Her2+ breast cancers had higher VEGFR2+ tumor vessel counts (p = 0.006). These data provide insights about tumor vascular density and vascular VEGFR2 expression in primary human breast cancer subtypes, especially Her2 positive breast cancer, and offer a pathobiologic hypothesis for the patterns of clinical response of breast cancer patients to newer anti-angiogenic therapies. Citation Format: Aejaz Nasir, Timothy R. Holzer, Michael Man, Laura E. Benjamin, Allen S. Melemed, Andrerw E. Schade. Differential expression of VEGFR2 Protein in HER2 positive primary human breast cancer: Potential relevance to newer anti-angiogenic therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4161. doi:10.1158/1538-7445.AM2015-4161

Placental Angiogenic Factors Are Associated With Maternal Thyroid Function and Modify hCG-Mediated FT4 Stimulation.

The thyroid has a high vascular density and this vascularity may be influenced by pregnancy-specific angiogenic factors. Proangiogenic placental growth factor (PlGF) and antiangiogenic soluble FMS-like tyrosine kinase-1 (sFlt1; a vascular endothelial growth factor [VEGF] and PlGF antagonist) are important pregnancy-specific angiogenesis regulators. We previously showed that fetal levels of sFlt1 and PlGF are associated with newborn thyroid function. However, the maternal thyroid may also be affected as PlGF and VEGF are secreted into the maternal circulation and cause a concomitant increase of sFlt1 to overcome adverse effects of angiogenesis overstimulation. Maternal sFlt1, PlGF, TSH, FT4, or human chorionic gonadotropin (hCG) levels were determined during early pregnancy (<18 wk) in 5517 women from the Generation R study. Analyses were adjusted for relevant covariates and interaction between hCG and angiogenic factors was investigated. Increasing levels of sFlt1 were associated with a decrease in FT4 and T4 (both P < .001), and an increased risk of subclinical hypothyroidism (odds ratio [OR] for high levels, 2.37; 95% CI, 1.16-4.83; P = .02) and isolated hypothyroxinemia (linear P = .02; OR, 3.05; 95% CI, 1.42-6.55; P = .004). Increasing levels of PlGF were associated with a decrease in TSH and FT4 levels (both P < .001), and an increased risk of isolated hypothyroxinemia (linear P = .002; OR, 1.77; 95% CI, 1.02-3.06; P = .04). High levels of hCG decreased the difference in FT4 between low and high sFlt1. In women with high PlGF levels, the hCG-mediated increase in FT4 levels was attenuated. sFlt1 and PlGF are novel determinants of maternal thyroid (dys)function during early pregnancy and the response of the maternal thyroid function to hCG stimulation. These data provide novel insights into the pregnancy specific thyroid function physiology and suggest that high levels of pro- and anti-angiogenic factors may be a risk factor for adverse pregnancy outcomes via their effects on maternal thyroid function.

Stress-mediated progression of solid tumors: effect of mechanical stress on tissue oxygenation, cancer cell proliferation, and drug delivery.

Oxygen supply plays a central role in cancer cell proliferation. While vascular density increases at the early stages of carcinogenesis, mechanical solid stresses developed during growth compress tumor blood vessels and, thus, drastically reduce not only the supply of oxygen, but also the delivery of drugs at inner tumor regions. Among other effects, hypoxia and reduced drug delivery compromise the efficacy of radiation and chemo/nanotherapy, respectively. In the present study, we developed a mathematical model of tumor growth to investigate the interconnections among tumor oxygenation that supports cancer cell proliferation, the heterogeneous accumulation of mechanical stresses owing to tumor growth, the non-uniform compression of intratumoral blood vessels due to the mechanical stresses, and the insufficient delivery of oxygen and therapeutic agents because of vessel compression. We found that the high vascular density and increased cancer cell proliferation often observed in the periphery compared to the interior of a tumor can be attributed to heterogeneous solid stress accumulation. Highly vascularized peripheral regions are also associated with greater oxygenation compared with the compressed, less vascularized inner regions. We also modeled the delivery of drugs of two distinct sizes, namely chemotherapy and nanomedicine. Model predictions suggest that drug delivery is affected negatively by vessel compression independently of the size of the therapeutic agent. Finally, we demonstrated the applicability of our model to actual geometries, employing a breast tumor model derived from MR images.

The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

Neurogenic niches in the brain: help and hindrance of the barrier systems.

In the developing central nervous system, most neurogenesis occurs in the ventricular and subventricular proliferative zones. In the adult telencephalon, neurogenesis contracts to the subependyma zone and the dentate gyrus (subgranular zone) of the hippocampus. These restricted niches containing progenitor cells which divide to produce neurons or glia, depending on the intrinsic and environmental cues. Neurogenic niches are characterized by a comparatively high vascular density and, in many cases, interaction with the cerebrospinal fluid (CSF). Both the vasculature and the CSF represent a source of signaling molecules, which can be relatively rapidly modulated by external factors and circulated through the central nervous system. As the brain develops, there is vascular remodeling and a compartmentalization and dynamic modification of the ventricular surface which may be responsible for the change in the proliferative properties. This review will explore the relationship between progenitor cells and the developing vascular and ventricular space. In particular the signaling systems employed to control proliferation, and the consequence of abnormal vascular or ventricular development on growth of the telencephalon. It will also discuss the potential significance of the barriers at the vascular and ventricular junctions in the influence of the proliferative niches.