VEGF Response Research Articles

Interaction of the human Prostacyclin receptor with the PDZ adapter protein PDZK1: Role in Endothelial Cell Migration and Angiogenesis

Prostacyclin is increasingly implicated in re‐endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high‐density lipoprotein (HDL) scavenger receptor class B, type 1 (SR‐B1) adapter protein PDZ domain‐containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. The interaction is constitutive, but may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP‐dependent protein kinase (PK)A‐phosphorylation of PDZK1 at Ser505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cAMP generation. Consistent with a role in re‐endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR‐B1, small interfering RNA (siRNA)‐targeted disruption of PDZK1 abolished cicaprost‐mediated endothelial responses but did not affect VEGF responses. The identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players within the vascular endothelium. Funded by SFI.

Pathogenesis of the Hantavirus Pulmonary Syndrome

Hantavirus pulmonary syndrome caused by hantaviruses in the Americas presents as a broad clinical spectrum ranging from brief febrile prodrome with only thrombocytopenia to rapidly progressive fulminant pulmonary edema and shock. This vascular leak syndrome confined almost exclusively to the lung is initiated by the noncytolytic infection of capillary endothelial cells. A number of pathogenic mechanisms have been proposed, including immune cell-mediated injury, cytokine-mediated injury and enhanced VEGF responses from intercellular junctions resulting from highly specific virus–integrin interactions. This review examines evidence for each of these potential mechanisms, with relevant references to its sister syndrome, hemorrhagic fever with renal syndrome, in Eurasia. Any mechanism or combination of mechanisms must be able to explain the massive pulmonary capillary leak at the severe extreme of the spectrum, a disease manifestation without parallel in clinical medicine.

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK1: role in endothelial cell migration and angiogenesis

Prostacyclin is increasingly implicated in re-endothelialization and angiogenesis but through largely unknown mechanisms. Herein the high-density lipoprotein (HDL) scavenger receptor class B, type 1 (SR-B1) adapter protein PDZ domain-containing protein 1 (PDZK1) was identified as an interactant of the human prostacyclin receptor (hIP) involving a Class I PDZ ligand at its carboxyl terminus and PDZ domains 1, 3, and 4 of PDZK1. Although the interaction is constitutive, it may be dynamically regulated following cicaprost activation of the hIP through a mechanism involving cAMP-dependent protein kinase (PK)A-phosphorylation of PDZK1 at Ser-505. Although PDZK1 did not increase overall levels of the hIP, it increased its functional expression at the cell surface, enhancing ligand binding and cicaprost-induced cAMP generation. Consistent with its role in re-endothelialization and angiogenesis, cicaprost activation of the hIP increased endothelial cell migration and tube formation/in vitro angiogenesis, effects completely abrogated by the specific IP antagonist RO1138452. Furthermore, similar to HDL/SR-B1, small interfering RNA (siRNA)-targeted disruption of PDZK1 abolished cicaprost-mediated endothelial responses but did not affect VEGF responses. Considering the essential role played by prostacyclin throughout the cardiovascular system, identification of PDZK1 as a functional interactant of the hIP sheds significant mechanistic insights into the protective roles of these key players, and potentially HDL/SR-B1, within the vascular endothelium.

Molecular targets of celastrol derived from Thunder of God Vine: Potential role in the treatment of inflammatory disorders and cancer

Identification of active constituents and their molecular targets from traditional medicine is an enormous opportunity for modern pharmacology. Celastrol is one such compound that was originally identified from traditional Chinese medicine (Thunder of God Vine) almost three decades ago and generally used for the treatment of inflammatory and auto-immune diseases. Celastrol has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. The anti-inflammatory effects of this triterpene have been demonstrated in animal models of different inflammatory diseases, including arthritis, Alzheimer’s disease, asthma, and systemic lupus erythematosus. This triterpene has also been found to inhibit the proliferation of a variety of tumor cells and suppress tumor initiation, promotion and metastasis in various cancer models in vivo. Celastrol’s ability to modulate the expression of pro-inflammatory cytokines, MHC II, HO-1, iNOS, NF-κB, Notch-1, AKT/mTOR, CXCR4, TRAIL receptors DR4 and DR5, CHOP, JNK, VEGF, adhesion molecules, proteasome activity, topoisomerase II, potassium channels, and heat shock response has been reported. This review describes the various molecular targets of celastrol, cellular responses to celastrol, and animal studies with celastrol in cancer and other inflammatory disorders.

Effects of acid–base balance and high or low intensity exercise on VEGF and bFGF

The purpose of the present study was to compare the acute hormonal response to a short-term high-intensity training (HIT) versus a high-volume endurance training (HVT) and to investigate the effects of acid-base status on cytokines involved in angiogenesis (VEGF and bFGF). Eleven subjects participated in three experimental trials. Two times subjects performed four 30-s "all-out" exercise bouts on a cycle ergometer separated by 5-min rest each, at which subjects either received bicarbonate (HIT (B)) or a placebo (HIT (P)) before the exercise. The third exercise trail consisted of a constant load exercise for 1 h at 50% peak power output (PPO). Venous blood samples were taken under resting conditions, 10, 60, and 240 min after each exercise condition to determine VEGF and bFGF serum concentrations. Capillary blood samples were taken to determine lactate concentrations and blood gas parameters. Mean pH values were significantly higher during HIT (B) compared to HIT (P). Serum VEGF concentration was significantly increased 10-min post-exercise in both HIT interventions. HVT showed no significant effects on VEGF levels. The diminished acidosis during HIT (B) had no effects on the VEGF response. There were no significant changes in bFGF in response to HIT or HVT. The present study suggests that HIT is a stimulus for exercise-induced VEGF secretion. These findings might be relevant for the arrangement of training, due to the fact that most of the training is often performed at low intensities possibly leading to an insufficient stimulus for VEGF secretion and angiogenesis.

Vascular Endothelial Growth Factor Increases Endothelial Nitric Oxide Synthase Transcription In Huvec Cells.

Although it is known that VEGF increases eNOS protein, the mechanisms responsible remain unclear. To determine if VEGF alters eNOS transcription, human umbilical vein endothelial cells were transfected with reporters under the control of the eNOS promoter and stimulated with VEGF165. VEGF significantly increased eNOS-mRNA after 2 hours exposure. VEGF significantly increased eNOS reporter activity as early as one hour (268±32%), but this increase returned to baseline after 6 hours. Using deletion constructs, the VEGF response region was initially localized to within the -722/-494 region. GMSA indicated that VEGF increased DNA binding to both a cAMP-like and AP1-like response elements. Site-specific mutations and heterologous constructs indicated that the site centered at AP1-like site was both necessary and sufficient to meditate VEGF transcriptional activation. These results indicate that VEGF rapidly activates eNOS transcription prior to a rise eNOS-mRNA, an effect mediated by a cis-trans interaction localized to an AP1-like site within the eNOS promoter.

VEGF Response To Exercise In Selectively Bred High-Aerobic Capacity Mice: Evidence for Genetic Influences?

VEGF Response To Exercise In Selectively Bred High-Aerobic Capacity Mice: Evidence for Genetic Influences?

Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor beta (PDGFRbeta) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-beta-dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRbeta-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions.

Genetic deletion of COX-2 diminishes VEGF production in mouse retinal Müller cells

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0–24 h. COX-2 protein and activity were assessed by western blot analysis and GC–MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 μM PGE 2, or PGE 2 + 5 μM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein ( p < 0.05) and activity ( p < 0.05), and VEGF production ( p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia ( p < 0.05). Of the prostanoids, PGE 2 was significantly increased by hypoxia ( p < 0.02). Exogenous PGE 2 significantly increased VEGF production by Müller cells ( p < 0.0039), and this effect was inhibited by H-89 ( p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE 2, signaling through the EP 2 and/or EP 4 receptor and PKA, mediates the VEGF response of Müller cells.

ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.

Exercise-induced VEGF transcriptional activation in brain, lung and skeletal muscle

Muscle VEGF expression is upregulated by exercise. Whether this VEGF response is regulated by transcription and/or post-transcriptional mechanisms is unknown. Hypoxia may be responsible: myocyte P(O2) falls greatly during exercise and VEGF is a hypoxia-responsive gene. Whether exercise induces VEGF expression in other organs important to acute physical activity is also unknown. To address these questions, we created a VEGF-Luciferase reporter mouse and measured VEGF transcription, mRNA and protein responses to (a) acute exercise and (b) short-term hypoxia (FI(O2) = 0.06) in brain (brainstem, cerebellum, cortex, hippocampus and striatum), muscle, lung, heart and liver. Exercise increased VEGF transcription, mRNA and protein in brain (hippocampus only), lungs and skeletal muscles, but not liver or heart. Hypoxia increased VEGF expression only in brain (cortex, hippocampus and striatum). New transcription appears to be a major exercise-induced regulatory step for increasing VEGF expression in muscle, lung and brain. Hippocampal VEGF expression was the only component of the exercise response recapitulated by hypoxia equivalent to the Everest summit.

Different roles of Foxo1 and Foxo3 in the control of endothelial cell morphology

Foxo1, a member of the Foxo subfamily of forkhead box transcription factors, is known to be essential for progression of normal vascular development in the mouse embryos. In the cultures of endothelial cells derived from embryonic stem cells, Foxo1-deficient endothelial cells exhibit an abnormal morphological response to vascular endothelial growth factor-A (VEGF-A), which is characterized by a lack of cell elongation, yet the molecular mechanisms governing endothelial cell morphology under angiogenic stimulation remain unknown. Here, we report that transforming growth actor-beta also induces endothelial cell elongation in collaboration with Foxo1 and VEGF-A. Furthermore, tetracycline-regulated induction of Foxo3, another member of the Foxo subfamily, into Foxo1-null endothelial cells failed to restore abnormal morphological response to VEGF-A at an early differentiation stage. In contrast, Foxo1 and Foxo3 exerted the same function at a late differentiation stage, i.e. enhancement of VEGF responsiveness and promotion of cell elongation. Our results provide evidence that endothelial cell morphology is regulated by several mechanisms in which Foxo1 and Foxo3 express distinct functional properties depending on differentiation stages.

Rapamycin reduces primary effusion lymphoma progression by targeting VEGF production and VEGF responses

Kaposi's sarcoma herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) typically presents as a malignant effusion in body cavities that disseminates to distant sites. There is a need for new therapies for PEL as most patients rapidly succumb in spite of high-dose chemotherapy. We examined the potential utility of Rapamycin for PEL treatment. We found that the downstream effectors of the mammalian target of Rapamycin (mTOR) p70S6k and S6 are constitutively phosphorylated in PEL cells, and that Rapamycin inhibits this constitutive p70S6k and S6 phosphorylation. Rapamycin reduces PEL proliferation but is not cytotoxic for PEL cells. Intraperitoneal injection of PEL cells in NOD/SCID mice causes experimental effusion lymphoma. Rapamycin delayed PEL development in this model, markedly reduced accumulation of ascites (p = 0.009), prevented formation of solid tumor masses, and a significantly extended mouse survival (P < 0.001). However, Rapamycin did not eradicate PEL in mice. We examined the mechanisms by which Rapamycin reduces PEL progression in this mouse model. Levels of VEGF, which promotes vascular permeability and is critical to the accumulation of body cavity fluids, were significantly reduced in ascites of Rapamycin-treated mice compared to controls (p = 0.009). Rapamycin inhibited VEGF-induced phosphorylation of VEGF receptor (R) 2 in endothelial cells and activation of the downstream effectors of VEGFR2 phosphorylation src and enos (endothelial nitric oxide synthase). Rapamycin did not alter KSHV genes transcription in PEL cells, and only insignificantly reduced levels of IL-10, the principal growth factor for PEL, in ascites of PEL-bearing mice. Reduction of VEGF secretion by PEL and impairment of endothelial cell responses to residual VEGF likely explain reduced accumulation of ascites in Rapamycin-treated mice. The failure of Rapamycin to significantly reduce IL-10 levels in PEL-bearing mice and to promote PEL cell death likely explain PEL persistence in mice treated with Rapamycin. The successful use of Rapamycin to reduce PEL effusion and disease progression by reducing VEGF secretion and endothelial cell responses to VEGF illustrates a novel application of mTOR inhibition that targets the tumor microenvironment rather than the tumor cells, and is applicable to the treatment of PEL and other malignancies characterized by ascites accumulation and increased vascular permeability.

Complexity of VEGF Responses in Skin Carcinogenesis Revealed through Ex Vivo Assays Based on a VEGF-A Null Mouse Keratinocyte Cell Line

Vascular endothelial growth factor (VEGF-A) is a critical player in cutaneous angiogenesis. However, the relative contribution of VEGF-A from different sources including epithelial and mesenchymal cells has not been fully characterized during skin repair and tumorigenesis. Moreover, the actual involvement of other vascular-specific acting molecules has remained elusive in part due to the masking and/or overlapping effects of VEGF-A. To shed light on these uncertainties we generated and characterized a clonal VEGF-null mouse keratinocyte cell line, through in vitro adenoviral gene transfer of Cre recombinase to VEGF-LoxP primary keratinocytes followed by repeated cell passaging under controlled conditions and cloning. In vitro and in vivo assays demonstrated that VEGF-null keratinocytes were nontumorigenic and expressed normal differentiation markers after calcium switch. Hras-induced tumorigenesis of immortalized VEGF-null keratinocytes upon subcutaneous injection was markedly reduced but not fully suppressed. However, the metastatic ability of Hras-transformed VEGF-null keratinocytes was abolished. These ex vivo approaches suggest the existence of VEGF-dependent and independent angiogenic stimuli in skin carcinogenesis. The VEGF-null mouse keratinocyte cell line arises as an important tool to assess the actual contribution of keratinocyte-derived VEGF with respect to other angiogenic factors in skin homeostasis and malignancy.

AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

Effect of diabetes mellitus on NGF and NGF‐receptor distribution in retinal ganglion cells and vascularity in adult rats

Abstract Purpose Recent studies reported that NGF exerts a protective action on cells of the visual system, including retinal cells, and that eye topical application of NGF can reach brain NGF‐responsive cells. The aim of the present study is to investigate the response of NGF, NGF‐receptor and VEGF in retinal cells in a rat model of DM. Methods Adult SD rats were obtained from local animal facilities, maintained in a 12:12‐hr light:dark cycle and had free access to tap water and food through out the experiments. Rats received a single intraperitoneal injection with 70mg/kg body weight of Streptozotocin (STZ) dissolved in PBS. Control rats received an equivalent volume of buffer solution. Animals were sacrificed with an overdose of Nembutal and pancreas, retina, optic nerve and lachrymal gland removed and used for biochemical, immunohistochemical, and molecular analysis. NGF was determined with “NGF Emax Immunoassay System” ELISA kit by Promega (USA). For histological and immunohistochemical analysis, eye globes were fixed in Bouin fluid. Twenty µm thick sections were cut with a cryostat at ‐20°C and immunostained for localization of NGF, NGF‐receptors, or vascular endothelial growth factor. Results DT lowers the presence of NGF in the whole retina and reduces the number of retinal cells, particularly in the retinal ganglion layer, and the presence of retinal vessels. NGF administration markedly reduces these deficits. Conclusion The present findings support the hypothesis of a NGF role in retinal cell physiopathology and suggest that eye NGF application might be useful to prevent and/o reducing retinopathy induced by DT. *V. Colafrancesco has a fellowship supported by Bietti Foundation, Roma

Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.

Heparan Sulfate in trans Potentiates VEGFR-Mediated Angiogenesis

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

VEGF as a predictive marker of rectal tumor response to preoperative radiotherapy

Neoadjuvant radiotherapy for rectal cancer may result in tumor downstaging or complete tumor regression leading to greater sphincter preservation. The identification of molecular predictive markers of tumor response to preoperative radiotherapy would provide an additional tool for selecting patients most likely to benefit from treatment. The aim of this study was to determine whether VEGF expression in preirradiation tumor biopsies is a useful predictive marker of tumor response in patients with rectal cancer undergoing preoperative radiotherapy. Immunohistochemistry for VEGF was performed on 59 preirradiation biopsies from patients with completely responsive (ypT0) or nonresponsive tumors after preoperative radiotherapy. VEGF positivity was evaluated using several scoring methods and the association between VEGF and tumor response was compared. The distribution of VEGF scores was obtained as well as the mean VEGF expression in the two response groups. The mean VEGF expression in nonresponsive tumors (NR) was significantly greater than in completely responsive tumors (CR) (P = 0.0035). Nearly half (47%) of all CR tumors had a VEGF expression of 10% or less. Eleven tumors were negative (0% immunoreactivity) for the protein and all of these (100%) were complete responders. Fifty-two percent of the NR tumors had VEGF scores of 80% or greater. The four scoring methods used to determine the association between VEGF and tumor response each produced significant results (P < 0.05). The results of this study indicate that VEGF assessed immunohistochemically from preirradiation tumor biopsies may be a useful marker of rectal tumor response to preoperative radiotherapy.