Proangiogenic cells (PACs) display surface markers and secrete angiogenic factors similar to those used by myelomonocytic cells, but, unlike myelomonocytic cells, PACs enhance neovascularization activity in experimental ischemic diseases. This study was performed to reveal the differential neovascularization activities of PACs compared with those of myelomonocytic cells. We cultured PACs and CD14(+)-derived macrophages (Macs) for 7 days. Most of the surface markers and cytokines in the two cell types were alike; the exceptions were KDR, β8 integrin, interleukin-8 and monocyte chemotactic protein-1. Unlike Macs, PACs significantly enhanced mesenchymal stem cell (MSC) transmigration. PACs and Macs increased neovascularization activity in an in vitro co-culture of human umbilical vein endothelial cells and MSCs and in an in vivo cotransplantation in Matrigel. However, the use of Macs resulted in inappropriately dilated and leaky vessels, whereas the use of PACs did not. We induced critical hindlimb ischemia in nude mice, and then transplanted PACs, Macs or vehicle into the mice. We obtained laser Doppler perfusion images weekly. At 2 weeks, mice treated with PACs showed significantly enhanced perfusion recovery in contrast to those treated with Macs. After day 7, when cells were depleted using a suicidal gene, viral thymidine kinase, to induce apoptosis of the cells in vivo by ganciclovir administration, we found that the improved perfusion was significantly abrogated in the PAC-treated group, whereas perfusion was not changed in the Mac-treated group. PACs caused an increase in healthy new vessels in in vitro and in vivo models of angiogenesis and enhanced long-term functional neovascularization activity in the hindlimb ischemia model, whereas Macs did not. Nevertheless, the angiogenic potential and long-term functional results for a specific cell type should be validated to confirm effectiveness and safety of the cell type for use in therapeutic angiogenesis procedures.

The tumor microenvironment is a critical regulator of cancer development and progression as well as treatment response and resistance in brain neoplasms. The available techniques for investigation, however, are not well suited for noninvasive in vivo characterization in humans. A total of 120 patients (59 females; 61 males) with newly diagnosed contrast-enhancing brain tumors (64 glioblastoma, 20 brain metastases, 15 primary central nervous system (CNS) lymphomas (PCNSLs), and 21 meningiomas) were examined with a previously established physiological MRI protocol including quantitative blood-oxygen-level-dependent imaging and vascular architecture mapping. Six MRI biomarker maps for oxygen metabolism and neovascularization were fused for classification of five different tumor microenvironments: glycolysis, oxidative phosphorylation (OxPhos), hypoxia with/without neovascularization, and necrosis. Glioblastoma showed the highest metabolic heterogeneity followed by brain metastasis with a glycolysis-to-OxPhos ratio of approximately 2:1 in both tumor entities. In addition, glioblastoma revealed a significant higher percentage of hypoxia (24%) compared to all three other brain tumor entities: brain metastasis (7%; p < 0.001), PCNSL (8%; p = 0.001), and meningioma (8%; p = 0.003). A more aggressive biological brain tumor behavior was associated with a higher percentage of hypoxia and necrosis and a lower percentage of remaining vital tumor tissue and aerobic glycolysis. The proportion of oxidative phosphorylation, however, was rather similar (17-26%) for all four brain tumor entities. Tumor microenvironment (TME) mapping provides insights into neurobiological differences of contrast-enhancing brain tumors and deserves further clinical cancer research attention. Although there is a long roadmap ahead, TME mapping may become useful in order to develop new diagnostic and therapeutic approaches.

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Vascular endothelial growth factor (VEGF) is a key angiogenic factors. It plays an important role in both physiologic and pathologic angiogenesis and increases permeability across the vessels. Using antibody phage display technology, we obtained a novel anti-VEGFA IgG, named as FD006. In this study, the pharmacological characteristics and efficacy of FD006 in corneal neovascularization (CoNV) were evaluated.

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide. Blindness can occur from retinal detachment caused by pathologic retinal angiogenesis into the vitreous, termed intravitreal neovascularization (IVNV). Although agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) are now used to treat IVNV, concerns exist regarding the identification of optimal doses of anti-VEGF for individual infants and the effect of broad VEGF inhibition on physiologic angiogenesis in external organs or in the retina of a preterm infant. Therefore, it is important to understand VEGF signaling in both physiologic and pathologic angiogenesis in the retina. In this manuscript, we review the role of receptors that interact with VEGF in oxygen-induced retinopathy (OIR) models that represent features of ROP pathology. Specifically, we discuss our work regarding the regulation of VEGFR2 signaling in retinal endothelial cells to not only reduce severe ROP but also facilitate physiologic retinal vascular and neuronal development.

Oxidized LDL (ox-LDL) is a key factor in atherogenesis. It is taken up by endothelial cells primarily by ox-LDL receptor-1 (LOX-1). To elucidate transcriptional responses, we performed microarray analysis on human coronary artery endothelial cells (HCAECs) exposed to small physiologic concentration of ox-LDL- 5 µg/ml for 2 and 12 hours. At 12 hours, cultures treated with ox-LDL exhibited broad shifts in transcriptional activity involving almost 1500 genes (>1.5 fold difference, p<0.05). Resulting transcriptome was enriched for genes associated with cell adhesion (p<0.002), angiogenesis (p<0.0002) and migration (p<0.006). Quantitative PCR analysis revealed that LOX-1 expression in HCAECs is at least an order of magnitude greater than the expression of other major ox-LDL specific receptors CD36 and MSR1. In keeping with the data on LOX-1 expression, pre-treatment of HCAECs with LOX-1 neutralizing antibody resulted in across-the-board inhibition of cellular response to ox-LDL. Ox-LDL upregulated a number of pro-angiogenic genes including multiple receptors, ligands and transcription factors and altered the expression of a number of genes implicated in both stimulation and inhibition of apoptosis. From a functional standpoint, physiologic concentrations of ox-LDL stimulated tube formation and inhibited susceptibility to apoptosis in HCAECs. In addition, ox-LDL exposure resulted in upregulation of miR-1974, miR-1978 and miR-21 accompanied with significant over-presentation of their target genes in the downregulated portion of ox-LDL transcriptome. Our observations indicate that ox-LDL at physiologic concentrations induces broad transcriptional responses which are mediated by LOX-1, and are, in part, shaped by ox-LDL-dependent miRNAs. We also suggest that angiogenic effects of ox-LDL are partially based on upregulation of several receptors that render cells hypersensitive to angiogenic stimuli.

To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye.

The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging.

A critical component of disease progression in rheumatoid arthritis (RA) involves neovascularization associated with pannus formation. 2-methoxyestradiol (2ME2) is a naturally occurring molecule with no known physiologic function, although at pharmacologic concentrations it has antiproliferative and antiangiogenic activities. We investigated the impact of orally administered 2ME2 on the initiation and development of proliferative synovitis using the anti-collagen monoclonal antibodies (CAIA) model.

The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions of the corneal and limbal epithelia in angiogenic and lymphangiogenic privilege, we designed five injury models involving debridement of different portions of the cornea and limbus and applied them to the dual-fluorescence reporter Prox1-GFP/Flt1-DsRed mouse, which permits in vivo imaging of blood and lymphatic vessels via fluorescence microscopy. Debridement of the whole cornea resulted in significant hemangiogenesis (HA) and lymphangiogenesis (LA), while that of the whole limbus yielded minimal corneal HA or LA. Following hemilimbal plus whole corneal debridement, corneal NV occurred only through the non-injured aspect of the limbus. Overall, these results suggest that the integrity of the corneal epithelium is important for (lymph)angiogenic privilege, whereas the limbus does not act as a physical or physiologic barrier to invading vessels. In CDh5-CreERT2VEGFR2lox/PGFD mice, conditional deletion of vascular endothelial growth factor receptor 2 in vascular endothelial cells abolished injury-induced HA and LA, demonstrating the utility of this transgenic mouse line for identifying important factors in the process of neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) in retinopathy of prematurity (ROP) raises concerns for premature infants because VEGF is essential for retinovascular development as well as neuronal and glial health. This study tested the hypothesis that endothelial cell-specific knockdown of VEGF receptor 2 (VEGFR2), or downstream STAT3, would inhibit VEGF-induced retinopathy without delaying physiologic retinal vascular development. We developed an endothelial cell-specific lentiviral vector that delivered shRNAs to VEGFR2 or STAT3 and a green fluorescent protein reporter under control of the VE-cadherin promoter. The specificity and efficacy of the lentiviral vector-driven shRNAs were validated in vitro and in vivo. In the rat oxygen-induced retinopathy model highly representative of human ROP, the effects of endothelial cell knockdown of VEGFR2 or STAT3 were determined on intravitreal neovascularization (IVNV), physiologic retinal vascular development [assessed as area of peripheral avascular/total retina (AVA)], retinal structure, and retinal function. Targeted knockdown of VEGFR2 or STAT3 specifically in retinal endothelial cells by subretinal injection of lentiviral vectors into postnatal day 8 rat pup eyes efficiently inhibited IVNV, and knockdown of VEGFR2 also reduced AVA and increased retinal thickness without altering retinal function. Taken together, our results support specific knockdown of VEGFR2 in retinal endothelial cells as a novel therapeutic method to treat retinopathy.

The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.

Background. The purpose of this experiment was to develop a peripheral nerve interface using cultured myoblasts within a scaffold to provide a biologically stable interface while providing signal amplification for neuroprosthetic control and preventing neuroma formation. Methods. A Regenerative Peripheral Nerve Interface (RPNI) composed of a scaffold and cultured myoblasts was implanted on the end of a divided peroneal nerve in rats (n = 25). The scaffold material consisted of either silicone mesh, acellular muscle, or acellular muscle with chemically polymerized poly(3,4-ethylenedioxythiophene) conductive polymer. Average implantation time was 93 days. Electrophysiological tests were performed at endpoint to determine RPNI viability and ability to transduce neural signals. Tissue samples were examined using both light microscopy and immunohistochemistry. Results. All implanted RPNIs, regardless of scaffold type, remained viable and displayed robust vascularity. Electromyographic activity and stimulated compound muscle action potentials were successfully recorded from all RPNIs. Physiologic efferent motor action potentials were detected from RPNIs in response to sensory foot stimulation. Histology and transmission electron microscopy revealed mature muscle fibers, axonal regeneration without neuroma formation, neovascularization, and synaptogenesis. Desmin staining confirmed the preservation and maturation of myoblasts within the RPNIs. Conclusions. RPNI demonstrates significant myoblast maturation, innervation, and vascularization without neuroma formation.