VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Coculture in vitro with endothelial cells induces cytarabine resistance of acute myeloid leukemia cells in a VEGF-A/VEGFR-2 signaling-independent manner.

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.

Interruption of vascular endothelial growth factor receptor 2 signaling induces a proliferative pulmonary vasculopathy and pulmonary hypertension.

Pulmonary arterial hypertension is a severe and progressive disease characterized by a pulmonary vascular remodeling process with expansion of collateral endothelial cells and total vessel occlusion. Endothelial cells are believed to be at the forefront of the disease process. Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptor, VEGF receptor-2 (VEGFR-2), play a central role in angiogenesis, endothelial cell protection, but also in the destabilization of endothelial barrier function. Therefore, we investigated the consequences of altered VEGF signaling in an experimental model, and looked for translational correlates of this observation in patients. We performed an endothelial cell-specific conditional deletion of the kinase insert domain protein receptor (kdr) gene, coding for VEGFR-2, in C57/BL6 mice (Kdr∆end) and held them in an environmental chamber with 10% FiO2 or under normoxia for 6 weeks. Kdr knockout led to a mild PH phenotype under normoxia that worsened under hypoxia. Kdr∆end mice exhibited a significant increase in pulmonary arterial wall thickness, muscularization, and VEGFR-3+ endothelial cells obliterating the pulmonary artery vessel lumen. We observed the same proliferative vasculopathy in our rodent model as seen in patients receiving anti-angiogenic therapy. Serum VEGF-a levels were elevated both in the experimental model and in humans receiving bevacizumab. Interrupted VEGF signaling leads to a pulmonary proliferative arteriopathy in rodents after direct ablative gene manipulation of Kdr. Histologically, similar vascular lesions can be observed in patients receiving anti-VEGF treatment. Our findings illustrate the importance of VEGF signaling for maintenance of pulmonary vascular patency.

Neuron-specific deletion of VEGF or its receptor Flk-1 impairs recognition memory.

Vascular endothelial growth factor (VEGF, also known as VEGF-A) is a pleiotropic factor which is expressed by neurons, astrocytes and perivascular macrophages, as well as endothelial cells, in the brain. Recently, VEGF signaling has been implicated in learning and memory, and several clinical and preclinical studies demonstrate that VEGF inhibitors induce cognitive impairment. However, the role of endogenous neuronal VEGF signaling in recognition memory remains unclear. Recently, we have developed mice with forebrain excitatory neuron-specific deletion of VEGF or its receptor, fetal liver kinase 1 (Flk-1) by crossing Camk2a-Cre mice with Vegfaflox/flox and Flk-1flox/flox mice, respectively. Using these conditional knockout mice, the present study addressed the influence of forebrain excitatory neuron-specific deletion of VEGF or Flk-1 on recognition memory in the novel object recognition test. The results show that both short-term (2 h) and long-term (24 h) recognition memory are impaired by neuron-specific deletion of either Flk-1 or VEGF. These findings indicate the physiological importance of endogenous neuronal VEGF-Flk-1 signaling in recognition memory. In addition, the current results also suggest that the impairment of neuronal VEGF-Flk-1 signaling can be a cause of anti-VEGF chemotherapy-induced cognitive impairment.

VEGF as a Trophic Factor for Müller Glia in Hypoxic Retinal Diseases.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR), leading causes of blindness, share a common retinal environment: hypoxia which is a major stimulator for the upregulation of vascular endothelial growth factor (VEGF), a cardinal pathogenic factor for the breakdown of blood-retina barrier (BRB). As a result of intensive studies on VEGF pathobiology, anti-VEGF strategy has become a major therapeutics for wet AMD and DR. To investigate the potential impact of anti-VEGF strategy on major retinal supporting cells, Müller glia (MG), we disrupted VEGF receptor-2 (VEGFR2) in MG with conditional knockout (CKO) and examined the effect of VEGFR2-null on MG viability and neuronal integrity in mice. VEGFR2 CKO mice demonstrated a significant loss of MG density in diabetes/hypoxia, which in turn resulted in accelerated retinal degeneration. These defects appear similar to the clinical characteristics in a significant portion of wet-AMD patients with long-term anti-VEGF therapies. In this article, we will discuss the potential relevance of these clinical characteristics to the critical role of VEGF signaling in MG viability and neuronal integrity in hypoxia.

LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage.

LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2-/- mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2-/- with Tal1-/- Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression.

Uncontrolled angiogenic precursor expansion causes coronary artery anomalies in mice lacking Pofut1.

Coronary artery anomalies may cause life-threatening cardiac complications; however, developmental mechanisms underpinning coronary artery formation remain ill-defined. Here we identify an angiogenic cell population for coronary artery formation in mice. Regulated by a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis, these angiogenic cells generate mature coronary arteries. The NOTCH modulator POFUT1 critically regulates this signaling axis. POFUT1 inactivation disrupts signaling events and results in excessive angiogenic cell proliferation and plexus formation, leading to anomalous coronary arteries, myocardial infarction and heart failure. Simultaneous VEGFR2 inactivation fully rescues these defects. These findings show that dysregulated angiogenic precursors link coronary anomalies to ischemic heart disease.Though coronary arteries are crucial for heart function, the mechanisms guiding their formation are largely unknown. Here, Wang et al. identify a unique, endocardially-derived angiogenic precursor cell population for coronary artery formation in mice and show that a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis is key for coronary artery development.

Endothelial marker-expressing stromal cells are critical for kidney formation.

Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins of renal endothelial cells and roles of the developing endothelia in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker-expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding-floxed Flk1 mice (Flk1fl/fl ) with Foxd1cre mice to generate Foxd1cre; Flk1fl/fl (Flk1ST-/- ) mice. We then performed FACsorting, histological, morphometric, and metabolic analyses of Flk1ST-/- vs. control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1ST-/- kidneys via flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1ST-/- mice, we found they had dilated peritubular capillaries. Three-dimensional reconstructions showed reduced ureteric branching and fewer nephrons in developing Flk1ST-/- kidneys vs. CONTROLS: Juvenile Flk1ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. Twenty-four-hour urine collections revealed that postnatal Flk1ST-/- mice had urinary-concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1-expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to nonautonomous-patterning defects in ureteric lineages.

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling.

RATIONALE: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

Defective angiogenesis delays thrombus resolution: a potential pathogenetic mechanism underlying chronic thromboembolic pulmonary hypertension.

OBJECTIVE: Restoration of patency is a natural target of vascular remodeling after venous thrombosis that involves vascular endothelial cells and smooth muscle cells, as well as leukocytes. Acute pulmonary emboli usually resolve <6 months. However, in some instances, thrombi transform into fibrous vascular obstructions, resulting in occlusion of the deep veins, or in chronic thromboembolic pulmonary hypertension (CTEPH). We proposed that dysregulated thrombus angiogenesis may contribute to thrombus persistence. APPROACH AND RESULTS: Mice with an endothelial cell-specific conditional deletion of vascular endothelial growth factor receptor 2/kinase insert domain protein receptor were used in a model of stagnant flow venous thrombosis closely resembling human deep vein thrombosis. Biochemical and functional analyses were performed on pulmonary endarterectomy specimens from patients with CTEPH, a human model of nonresolving venous thromboembolism. Endothelial cell-specific deletion of kinase insert domain protein receptor and subsequent ablation of thrombus vascularization delayed thrombus resolution. In accordance with these findings, organized human CTEPH thrombi were largely devoid of vascular structures. Several vessel-specific genes, such as kinase insert domain protein receptor, vascular endothelial cadherin, and podoplanin, were expressed at lower levels in white CTEPH thrombi than in organizing deep vein thrombi and organizing thrombi from aortic aneurysms. In addition, red CTEPH thrombi attenuated the angiogenic response induced by vascular endothelial growth factor. CONCLUSIONS: In the present work, we propose a mechanism of thrombus nonresolution demonstrating that endothelial cell-specific deletion of kinase insert domain protein receptor abates thrombus vessel formation, misguiding thrombus resolution. Medical conditions associated with the development of CTEPH may be compromising early thrombus angiogenesis.

Genetic reduction of vascular endothelial growth factor receptor 2 rescues aberrant angiogenesis caused by epsin deficiency.

OBJECTIVE: We previously showed that endothelial epsin deficiency caused elevated vascular endothelial growth factor receptor 2 (VEGFR2) and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and reduced tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. APPROACH AND RESULTS: A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages showed that deletion of epsins caused defective embryonic angiogenesis and retarded embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (ECs) from Epn1(fl/fl); Epn2(-/-); iCDH5 Cre (EC-iDKO) and Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre (EC-iDKO-Flk(fl/+)) mice demonstrated that VEGFR2 reduction in epsin-depleted cells was sufficient to restore normal VEGF signaling, EC proliferation, EC migration, and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. CONCLUSIONS: Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate that epsins may be a potential therapeutic target in conditions in which tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.

Down-regulation of Gab1 inhibits cell proliferation and migration in hilar cholangiocarcinoma.

Hilar cholangiocarcinoma is a highly aggressive malignancy originating from the hilar biliary duct epithelium. Due to few effective comprehensive treatments, the prognosis of hilar cholangiocarcinoma is poor. In this study, immunohistochemistry was first used to detect and analyze the expression of Gab1, VEGFR-2, and MMP-9 in hilar cholangiocarcinoma solid tumors and the relationships to the clinical pathological features. Furthermore, Gab1 and VEGFR-2 siRNA were used to interfere the hilar cholangiocarcinoma cell line ICBD-1 and then detect the PI3K/Akt signaling pathway, MMP-9 levels and malignant biological behaviors of tumor cells. The data showed that 1. Gab1, VEGFR-2, and MMP-9 were highly expressed and positively correlated with each other in hilar cholangiocarcinoma tissues, which were related to lymph node metastasis and differentiation. 2. After Gab1 or VEGFR-2 siRNA interference, PI3K/Akt pathway activity and MMP-9 levels were decreased in ICBD-1 cells. At the same time, cell proliferation decreased, cell cycle arrested in G1 phase, apoptosis increased and invasion decreased. These results suggest that the expression of Gab1, VEGFR-2, and MMP-9 are significantly related to the malignant biological behavior of hilar cholangiocarcinoma. Gab1 regulates growth, apoptosis and invasion through the VEGFR-2/Gab1/PI3K/Akt signaling pathway in hilar cholangiocarcinoma cells and influences the invasion of tumor cells via MMP-9.

[KDR silencing supresses the tumor growth of prostate cancer cell line PC-3 in nude mice].

OBJECTIVE: To study the change in the tumor growth of prostate cancer cell line PC-3 in nude mice xenografts after kinase domain receptor (KDR) silencing by RNA interference. METHODS: A total of 15 5-week-old male nude mice were randomly divided into normal PC-3 cell group (negative control), RNA interference group and pSilencer3.1-NC group, with 5 mice in every group. The nude mice were respectively treated with subcutaneous injection of 0.5 mL (2.0×10(7);/mL) normal PC-3 cells, and the same volume of PC-3 cells transfected with pSilencer3.1-KDR and pSilencer3.1-NC vectors, respectively. By measuring the tumor volumes every 3 d and the tumor weights after 4 weeks, we recorded tumor formation rate, tumor growth rate and mean tumor weight. The expression of KDR at both mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. RESULTS: Tumor growth was significantly slower in the pSilencer3.1-KDR group than in the negative control group and the pSilencer3.1-NC group. After 4 weeks, the mean volume of tumor in the pSilencer3.1-KDR group was significantly smaller than that in the other two groups (0.28 cm(3); vs 0.715 cm(3); and 0.721 cm(3);, P<0.01), so was the mean weight of tumor (0.14 g vs 0.635 g and 0.648 g, P<0.01). In addition, KDR mRNA and protein expressions significantly decreased. CONCLUSION: The tumor growth in nude mice xenografts can be efficiently inhibited by KDR silencing mediated by RNAi, so the suppression of KDR expression might be a promising strategy for the treatment of human prostate cancer.

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling.

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis.

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.

Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth.

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).

No functional and transductional significance of specific neuropilin 1 siRNA inhibition in colon carcinoma cell lines lacking VEGF receptor 2.

Neuropilins are membrane proteins that mediate effects on tumor cells directly and indirectly by affecting angiogenesis. Recent findings indicate that neuropilin 1 (NRP1) and the associated tyrosine kinase vascular endothelial growth factor receptor 2 (VEGFR2) play a regulatory role in developmental angiogenesis as well as in tumor angiogenesis. NRP1 and VEGFR2 might play a role in colon carcinogenesis and development of metastases. The significance of NRP1 expression in colon cancer seems to be controversial. Therefore, we aimed to distinguish between different expression patterns of signalling cascades in human colon carcinoma cell lines in order to analyze the role of NRP1 in tumorigenesis. We analyzed the biological significance of NRP1 in respect to VEGFR, EGFR, neuropilin and their ligands by RT-PCR and western blot with functional knock-out of NRP1 in different colon adenocarcinoma cell lines. There was no expression of VEGFR2 in tumor cell lines. There were cells that expressed: i) only NRP1 (HT-29, LS174T), ii) NRP2 (Colo320) or iii) both (SW480, LoVo). Cells without NRP1 expression strongly expressed EGFR but only when NRP2 was co-expressed. Inhibition of NRP1 expression by RNA interference did not alter growth characteristics in soft agar experiments. Furthermore, there were no differences in intracellular signalling pathways (ERK1/2 or AKT) in NRP1 inhibited cells. In ex vivo transfer experiments animals with tumors from siRNA-NRP1 transfected cells showed no significant inhibition of tumor growth compared to siRNA control. In conclusion, our results question the role of NRP1 function in VEGFR2 negative colon adenocarcinoma cells. NRP1 seems to have no detectable effect on proliferation or migration nor does it induce any changes in intracellular signalling pathways without the expression of VEGFR2. According to our data, further studies are needed to analyze the therapeutic relevance of NRP1 inhibition in vivo.

Vascular endothelial growth factor receptor-1 regulates postnatal angiogenesis through inhibition of the excessive activation of Akt.

Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs "fine tuning" of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1(+/-) mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1(+/-) Akt1(+/-) mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.