VEGF Receptor Flk-1 Research Articles

Neural progenitor cell implants in the lesioned medial longitudinal fascicle of adult cats regulate synaptic composition and firing properties of abducens internuclear neurons.

Transplants of neural progenitor cells (NPCs) into the injured CNS have been proposed as a powerful tool for brain repair, but, to date, few studies on the physiological response of host neurons have been reported. Therefore, we explored the effects of NPC implants on the discharge characteristics and synaptology of axotomized abducens internuclear neurons, which mediate gaze conjugacy for horizontal eye movements. NPCs were isolated from the subventricular zone of neonatal cats and implanted at the site of transection in the medial longitudinal fascicle of adult cats. Abducens internuclear neurons of host animals showed a complete restoration of axotomy-induced alterations in eye position sensitivity, but eye velocity sensitivity was only partially regained. Analysis of the inhibitory and excitatory components of the discharge revealed a normal re-establishment of inhibitory inputs, but only partial re-establishment of excitatory inputs. Moreover, their inhibitory terminal coverage was similar to that in controls, indicating that there was ultimately no loss of inhibitory synaptic inputs. Somatic coverage by synaptophysin-positive contacts, however, showed intermediate values between control animals and animals that had undergone axotomy, likely due to partial loss of excitatory inputs. We also demonstrated that severed axons synaptically contacted NPCs, most of which were VEGF immunopositive, and that abducens internuclear neurons expressed the VEGF receptor Flk1. Together, our results suggest that VEGF neurotrophic support might underlie the increased inhibitory-to-excitatory balance observed in the postimplant cells. The noteworthy improvement of firing properties of injured neurons following NPC implants indicates that these cells might provide a promising therapeutic strategy after neuronal lesions.

VEGFA-dependent and -independent pathways synergise to drive Scl expression and initiate programming of the blood stem cell lineage in Xenopus.

The first haematopoietic stem cells share a common origin with the dorsal aorta and derive from putative adult haemangioblasts in the dorsal lateral plate (DLP) mesoderm. Here we show that the transcription factor (TF) stem cell leukaemia (Scl/Tal1) is crucial for development of these adult haemangioblasts in Xenopus and establish the regulatory cascade controlling its expression. We show that VEGFA produced in the somites is required to initiate adult haemangioblast programming in the adjacent DLP by establishing endogenous VEGFA signalling. This response depends on expression of the VEGF receptor Flk1, driven by Fli1 and Gata2. Scl activation requires synergy between this VEGFA-controlled pathway and a VEGFA-independent pathway controlled by Fli1, Gata2 and Etv2/Etsrp/ER71, which also drives expression of the Scl partner Lmo2. Thus, the two ETS factors Fli1 and Etv6, which drives the VEGFA expression in both somites and the DLP, sit at the top of the adult haemangioblast gene regulatory network (GRN). Furthermore, Gata2 is initially activated by Fli1 but later maintained by another ETS factor, Etv2. We also establish that Flk1 and Etv2 act independently in the two pathways to Scl activation. Thus, detailed temporal, epistatic measurements of key TFs and VEGFA plus its receptor have enabled us to build a Xenopus adult haemangioblast GRN.

VEGF modulates NMDA receptors activity in cerebellar granule cells through Src-family kinases before synapse formation

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

Hsp90 and angiogenesis in bone disorders—lessons from the avian growth plate

Thiram-induced tibial dyschondroplasia (TD) and vitamin-D deficiency rickets are avian bone disorders of different etiologies characterized by abnormal chondrocyte differentiation, enlarged and unvascularized growth plates, and lameness. Heat-shock protein 90 (Hsp90) is a proangiogenic factor in mammalian tissues and in tumors; therefore, Hsp90 inhibitors were developed as antiangiogenic factors. In this study, we evaluated the association between Hsp90, hypoxia, and angiogenesis in the chick growth plate. Administration of the Hsp90 inhibitor to TD- and rickets-afflicted chicks at the time of induction resulted in reduction in growth-plate size and, contrary to its antiangiogenic effect in tumors, a major invasion of blood vessels occurred in the growth plates. This was the result of upregulation of the VEGF receptor Flk-1, the major rate-limiting factor of vascularization in TD and rickets. In addition, the abnormal chondrocyte differentiation, as characterized by collagen type II expression and alkaline phosphatase activity, and the changes in hypoxia-inducible factor-1α (HIF-1α) in both disorders were restored. All these changes resulted in prevention of lameness. Inhibition of Hsp90 activity reduced growth-plate size, increased vascularization, and mitigated lameness also in TD chicks with established lesions. In summary, this is the first reported demonstration of involvement of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the antiangiogenic effect of Hsp90 inhibitors observed in mammals, inhibition of Hsp90 activity in the unvascularized TD- and rickets-afflicted chicks resulted in activation of the angiogenic switch and reinstated normal growth-plate morphology.

Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation During Tissue Repair by Altering Gene Expression and Is Inhibited by Aspirin.

Abstract 3055Poster Board II-1031Fibrin deposition triggers an injury response that involves the migration of inflammatory cells, formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TGM2) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TGM2 is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGFbeta-1) and expressed during tissue injury. In this study, we investigated how TGM2 modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin) implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TGM2 into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p<0.001), an effect similar in magnitude to 25 ng/ml of TGFbeta1 administered in the F-ZC. The pro-healing effect of TGM2 was associated with a 2-fold increase in microvessel density in granulation tissue at day 6 of wound healing response (p<0.001). As a negative control, inactive recombinant C277A/TGM2 mutant did not exhibit increased wound healing response or proangiogenic effect. The data suggested that TGM2 enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TGM2 enhanced wound healing were 1) angiogenesis and 2) deposition of ECM. To investigate TGM2-induced angiogenesis-related gene expression, total RNAs were isolated from control- and TGM2-treated F-ZCs (at Day 6). Biotin-labeled cDNA probes were synthesized, and hybridized to nylon membranes containing angiogenesis-related gene arrays (Superarray, MD). The signals were detected using streptavidin-peroxidase and quantitated. We identified increased expression of VEGF receptors Flk-1 (2-fold), Flt1 and neuropilin (1.4-fold), angiopoietin-1 (2-fold) and ephrin B2 (1.8-fold). There were decreased levels (5-fold) of matrix metalloproteinases (MMPs) and increased TGFbeta-1 receptors (1.5-fold) and connective tissue growth factor (CTGF)(1.4-fold) levels. The gene expression profile suggests that TGM2 promotes angiogenesis and enhances deposition of ECM.We then investigated whether Aspirin (Acetylsalicylic Acid, ASA) a potent anti-inflammatory agent would inhibit TGM2. ASA and another chemical acetylating agent, sulfosuccinimidyl acetate (SNA), were used to investigate whether acetylation would alter the crosslinking activity of TGM2. We found acetylation by either SNA or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys468 and Lys663. Molecular modeling indicates that these Lys residues play an important role in the conformation change that occurs in TGM2 from a closed-to-open shape, i.e. inactive-to-active, transitions. In conclusion, we show that TGM2-fibrin crosslinking accelerates angiogenesis and promotes ECM deposition. This suggests that TGM2-fibrin interactions mediates outside-in signaling events that aides wound healing. Furthermore aspirin can acetylate and inhibit critical residues in TGM2 that regulate TGM-2 function. DisclosuresNo relevant conflicts of interest to declare.

The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation ofVegf

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

Human umbilical vascular endothelial cells express estrogen receptor beta (ERβ) and progesterone receptor A (PR-A), but not ERα and PR-B

Several reports deal with possible effects of female sex hormones on human umbilical vein endothelial cells (HUVEC) including elasticity, activation of plasma membrane Na+/H+ exchange, VEGF receptor Flk-1/KDR and many others. In contrast to those findings, some publications pointed out that HUVEC lack expression of both the estrogen receptor (ER) and/or the progesterone receptor (PR). Because the majority of these investigations were carried out at a time period, when only one ER and one PR was known, the aim of this study was the systematic analysis of ERalpha and ERbeta as well as PR-A and PR-B expression in HUVEC with specific monoclonal antibodies by immunocytochemistry and quantitative RT-PCR (TaqMan). As a result, we could show that HUVEC lack ERalpha but express ERbeta. The expression of ERbeta could be significantly upregulated with 17beta-estradiol on mRNA and protein level. In addition, HUVEC express PR-A but not PR-B. PR-A expression could be significantly upregulated with progesterone, again on mRNA and protein level. We conclude that estrogenic effects on HUVEC are mediated via the ERbeta and gestagens act via the PR-A pathway.

Roles of the endogenous VEGF receptors flt-1 and flk-1 in astroglial and vascular remodeling after brain injury

Following trauma to the brain significant changes occur in both the astroglial and vascular components of the neuropil. Angiogenesis is required to re-establish metabolic support and astrocyte activation encompasses several functions including scar formation and the production of growth factors. VEGF has seminal involvement in the process of brain repair and is upregulated during many pathological events. VEGF signaling is regulated mainly through its two primary receptors: flk-1 (KDR/VEGF-R2) is expressed on vascular endothelium and some neurons and flt-1 (VEGF-R1) in the CNS, is expressed predominantly by activated astrocytes. Using an injury model of chronic minipump infusion of neutralizing antibodies (NA) to block VEGF receptor signaling, this study takes advantage of these differences in VEGF receptor distribution in order to understand the role the cytokine plays after brain injury. Infusion of NA to flk-1 caused a significant decrease in vascular proliferation and increased endothelial cell degeneration compared to control IgG infusions but had no effect on astrogliosis. By contrast infusion of NA to flt-1 significantly decreased astroglial mitogenicity and scar formation and caused some increase in endothelial degeneration. Neutralization of the flt-1 receptor function, but not flk-1, caused significant reduction in the astroglial expression of the growth factors, CNTF and FGF by 7days. These data suggest that after CNS injury, endogenous VEGF upregulation (by astrocytes) induces angiogenesis and, by autocrine signaling, increases both astrocyte proliferation and facilitates expression of growth factors. It is likely that VEGF plays an important role in aspects of astroglial scar formation.

Angiogenesis in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Impaired placental angiogenesis during early pregnancy may result in placental defects that adversely affect development of nuclear-transfer (NT) embryos later in pregnancy. These experiments were designed to quantify and compare development of placental microvasculature and expression of genes associated with angiogenesis, including members of the VEGF and angiopoietin (Ang) families, in maternal and embryonic placental tissues of day 30 bovine concepti derived from NT or in vitro fertilization (IVF) followed by in vivo development to the blastocyst stage in the sheep oviduct. Microvascular volume density (MVD) within the caruncular tissues, as determined using Periodic Acid-Schiff's staining as well as immunohistochemical staining for von Willebrand's factor, was not different between NT- and IVF- derived pregnancies. Expression of genes implicated in angiogenic mechanisms, including VEGF-A and -C, placental growth factor (PlGF), VEGF receptors (Flt-1, Flk-1, and Flt-4), angiopoietin-1 (Ang1), Ang2, Tie1, Tie2, and hypoxia-inducible factor (HIF), were determined. In chorio-allantoic membranes, levels of PlGF transcripts were significantly lower in NT- than IVF-derived tissues (p<0.05), whereas HIF-1alpha transcription in chorio-allantoic membranes of cloned concepti was higher at p<0.10. Caruncular expression of HIF-1alpha and Ang1 also was increased in NT-derived pregnancies at p <or= 0.10. Immunohistochemical staining of caruncular tissues for VEGF-A and the Flt-1 receptor revealed few differences in protein expression between NT- and IVF-derived pregnancies. These results indicate that expression of most angiogenic factors at day 30 of gestation is not altered as a result of the NT procedure; however, given reports of impaired placental vascular development in NT-derived bovine embryos, perturbations in angiogenesis may occur subsequently during early placental development and throughout gestation. Elevated expression of the HIF-1alpha gene in maternal and chorio-allantoic tissues of cloned concepti may suggest a generalized hypoxic condition in early placental tissues of NT-derived concepti, which could adversely affect subsequent development of the placenta.

VEGF receptor Flk-1 plays an important role in c-kit expression in adipose tissue derived stem cells

It is known that c-kit + cells are increased in heart after infarction. The exact origins of the cardiac c-kit + cells remain to be determined. We asked whether adipose tissue could be a potential source of c-kit + cells. Our data show that the number of c-kit + cells increased in adipose tissue derived stem cells when cultured with conditioned medium from neonatal cardiomyocytes grown under serum deprivation and hypoxia condition. We also found that VEGF receptor Flk-1 is involved in c-kit up regulation via ERK-mediated pathway.

Inhibition of tumor angiogenesis by cell-permeable dominant negative SOX18 mutants

Angiogenesis play a key roles in tumor growth, invasion and metastasis, and has become an attractive target for anticancer drug development. Though a number of anti-angiogenic agents had entered clinical trials, few of them could reproduce the spectacular results in cancer patients as that had been seen in pre-clinical tumor models. Therefore, exploring novel anti-angiogenic agents is highly deserved. SOX18, a member of the Sry-related HMG box-containing family of transcription factors, is expressed transiently in endothelial cells during the development of blood vessels. And mutations resulting in expression of dominant negative SOX18 have been shown to severely impair the vascular development. Recent research demonstrated that SOX18 is expressed during the initial steps of tumor vascularization and involved in regulation of the expression of the VEGF receptor Flk-1 and the vascular cell adhesion molecule-1 (VCAM-1). Moreover, allograft tumor growth in mice heterozygous for Ra(Op) (RaOp mice) which express a dominant negative mutant form of SOX18 (SOX18RaOp) that does not interact effectively with the endothelial partner protein MEF2C, was dramatically slower than that of wild-type mice. In this article, we postulate that recombinant cell-permeable dominant negative SOX18 mutants, prepared by fusion with protein transduction domains, would inhibit tumor angiogenesis with high efficiency by impairing endothelial tube formation. If the hypothesis was proved to be practical, the fusion proteins would show promise as single anti-angiogenic agents in cancer therapy.

A Common Progenitor at the Heart of Development

Formation of the heart requires the coordinated functions of cardiac myocytes, smooth muscle cells, endothelial cells, and connective tissue elements. Several recent studies now reveal that these different cell types arise from a common progenitor (). These findings raise interesting questions about the lineage relationships of cardiovascular progenitor cell populations and suggest possibilities for cardiac repair in both congenital and acquired heart disease.

Angiogenic Cytokine Induction in Lung Injury Response to 2‐Chloroethyl Ethyl Sulfide, a Mustard Gas Analog

We reported earlier that guinea pigs exposed to 2-chloroethyl ethyl sulfide (CEES) as a mustard gas analog, accumulate inflammatory cytokines TNF-α,IL-1αINFγand chemokines, Eotaxin and MIP-1δin the lung, at higher levels within 1 h. Since angiogenesis may play a role, in inflammation and pathophysiology of tissue remodeling, we evaluated the early induction profile of angiogenic cytokines and selected target genes using cytokine antibody arrays. VEGF and bFGF levels showed 1.5-fold increase within 1 h of exposure. However, while VEGF remained high after one day, bFGF level was decreased. We detected 2-fold increase of numerous pro-angiogenic cytokines, members of FGF and TGF-β family, PDGF-BB, interleukins (IL-1, IL-4) and TNF-α within 1hr, most of which induce expression of VEGF, a potent angiogenic factor. Essential mediators in angiogenesis and tissue remodeling such as TSP, TIMP2 (1hr.) and uPAR (1 day) were increased by 1.7-fold. Increase in VEGF receptors Flk-1 and Flt-1and MMP-2 were also detected by Western blot analysis in 1 day. Our results suggest a disruption in vascular quiescence maintained by the balance between stimulators and inhibitors of angiogenesis as early as 1hr. post CEES. Regulated by various proinflamatory cytokines, VEGF may thus play an essential role in inducing chronic inflammation, fibrosis, and cancer following lung injury. (Supported by US Army Grant DAMD 17-03-2-0054)

Flk1 expression: promiscuity revealed

Comment on Ema et al, page [111][1] New evidence shows that Flk1 (VEGFR-2) is broadly and transiently expressed in multipotent mesoderm during development, indicating that Flk1 expression does not uniquely define the hematovascular lineage. Although expression of the VEGF receptor Flk1 (VEGFR-2)

Increased Vascular Endothelial Growth Factor Expression But Impaired Vascular Endothelial Growth Factor Receptor Signaling in the Myocardium of Type 2 Diabetic Patients With Chronic Coronary Heart Disease

The aim of the present study was to evaluate the expression and the activity of vascular endothelial growth factor (VEGF) in the hearts of diabetic patients with chronic coronary heart disease (CHD). Diabetes is characterized by a decreased collateral vessel formation in response to coronary ischemic events, although the role of VEGF in human diabetic macroangiopathy has not been fully investigated. Biopsies of left ventricular (LV) myocardium were obtained from 10 patients with type 2 diabetes and 10 non-diabetic patients with chronic CHD, all undergoing surgical coronary revascularization. Right ventricle myocardial samples taken from normal hearts were used as control specimens. Vascular endothelial growth factor and VEGF-receptors (flt-1 and flk-1) were evaluated by Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Akt and endothelial nitric oxide synthase (eNOS) protein expression and their phosphorylated forms were also evaluated by Western blot. Vascular endothelial growth factor, flt-1, and flk-1 messenger ribonucleic acid (mRNA) and protein expressions were increased in non-diabetic patients with CHD compared with control subjects. Remarkably, in diabetic patients, VEGF mRNA and protein levels were significantly higher, whereas flt-1, flk-1 mRNA, and protein were lower when compared with non-diabetic patients. Interestingly, phospho-flk-1 was reduced in diabetic patients compared with non-diabetic patients. As a consequence, Akt phosphorylation, eNOS protein and its phosphorylated form were significantly higher in the samples from non-diabetic patients compared with diabetic patients. Chronic CHD in diabetic patients is characterized by an increased VEGF myocardial expression and a decreased expression of its receptors along with a down-regulation of its signal transduction. The latter could be partially responsible for the reduced neoangiogenesis in diabetic patients with ischemic cardiomyopathy.

The Vascular-Ablative Agent VEGF121/rGel Inhibits Pulmonary Metastases of MDA-MB-231 Breast Tumors

VEGF121/rGel, a fusion protein composed of the growth factor VEGF121 and the recombinant toxin gelonin (rGel), targets the tumor neovasculature and exerts impressive cytotoxic effects by inhibiting protein synthesis. We evaluated the effect of VEGF121/rGel on the growth of metastatic MDA-MB-231 tumor cells in SCID mice. VEGF121/rGel treatment reduced surface lung tumor foci by 58% compared to controls (means were 22.4 and 53.3, respectively; P < .05) and the mean area of lung colonies by 50% (210 ± 37 m2vs 415 ± 10 m2 for VEGF121/rGel and control, respectively; P < .01). In addition, the vascularity of metastatic foci was significantly reduced: (198 ± 37 vs 388 ± 21 vessels/mm2 for treated and control, respectively). Approximately 62% of metastatic colonies from the VEGF121/rGel-treated group had fewer than 10 vessels per colony compared to 23% in the control group. The VEGF receptor Flk-1 was intensely detected on the metastatic vessels in the control but not in the VEGF121/rGel-treated group. Metastatic foci present in lungs had a three-fold lower Ki-67 labeling index compared to control tumors. Thus, the antitumor vascular-ablative effect of VEGF121/rGel may be utilized not only for treating primary tumors but also for inhibiting metastatic spread and vascularization of metastases.

Hypoxia-Independent Regulation of Hepatic Erythropoietin Production by Vascular Endothelial Growth Factor.

Erythropoietin (Epo) is an essential regulator of red blood cell production. While adult Epo synthesis occurs almost exclusively in kidney, extrarenal Epo synthesis has been described in liver and brain in response to hypoxia or anemia. We have used adenoviruses to express soluble ectodomains of the VEGF receptors (sVEGFRs) Flk1 and Flt1 in the circulation of mice, achieving high-level VEGF sequestration, potent conditional ablation of VEGF function in adult animals, and inhibition of tumor growth and angiogenesis (Kuo et al. PNAS (2001). 98: 4605). Intriguingly, adenoviral expression of soluble Flk1 or Flt1 ectodomains, or a recently described soluble receptor VEGF Trap, is associated with profound stimulation of erythropoiesis and polycythemia, with hematocrit as high as 75% from basal levels of ~45%, and elevation of plasma Epo levels. sVEGFR expression results in a profound >100-fold induction of hepatic erythropoietin synthesis while renal Epo production is completely repressed. This effect is specific for adenoviruses expressing soluble VEGF receptors and not other receptor ectodomains, does not require liver transduction or hypoxia, and is not affected by Ad Cre-induced deletion of the hepatocyte HIF-1a gene in HIF-1aflox/flox mice. Selective inhibition of hepatocyte-produced VEGF in Ad Cre-treated VEGFflox/flox mice is sufficient to re-activate hepatocyte Epo production, likely via hepatocyte × endothelium crosstalk. These data suggest VEGF may function physiologically to repress hepatic Epo synthesis, while VEGF blockade leads to de-repression of hepatic Epo production via a novel hypoxia- and HIF-1a-independent pathway.

Tissue Transglutaminase Enhances Fibrin-Dependent Angiogenesis and Extracellular Matrix Formation by Altering Gene Expression during Wound Healing.

Fibrin deposition triggers an injury response that involves the migration of inflammatory cells formation of new blood vessels and the synthesis of extracellular matrix (ECM). Tissue transglutaminase (TTG) is a calcium dependent enzyme that covalently crosslinks a wide variety of ECM proteins producing a protease resistant matrix. TTG is secreted by inflammatory and endothelial cells, involved in activating transforming growth factor beta-1 (TGF beta-1) and expressed during wound healing response. In this study, we investigated how TTG modulated fibrin-dependent wound healing and the associated angiogenic response. We used an animal model consisting of fibrin Z-chambers (F-ZC, dual porous plexiglass chambers containing fibrin), implanted into the subcutaneous tissue of rats and harvested subsequently for quantitative assessment of granulation tissue formation (wound healing) and microvessel density (angiogenesis). We found that local administration of recombinant TTG into F-ZC resulted in a dose-dependent, 2-fold increase in granulation tissue thickness by day 6 of wound healing (p<0.001), an effect similar in magnitude to 25 ng/ml of TGFbeta1 administered in the F-ZC. The pro-healing effect of TTG was associated with a 2-fold increase in microvessel density in granulation tissue at day 6 of wound healing response (p<0.001). As a negative control, inactive recombinant TTG mutant did not exhibit increased wound healing response or pro-angiogenic effect. The data suggested that TTG enhanced the transition from the inflammatory stage of wound healing to proliferation stage. The two areas where TTG enhanced wound healing were 1) angiogenesis and 2) deposition of matrix. To investigate TTG-induced gene expression, total RNAs were isolated from control- and TTG-treated F-ZCs (at Day 6) using Trizol reagent (Invitrogen, CA). Biotin-labeled cDNA probes were synthesized, and hybridized to nylon membranes containing angiogenesis-related gene arrays (Superarray, MD). The signals were detected using streptavidin-peroxidase and quantitated using Superarray's software. We identified increased expression of VEGF receptors Flk-1, Flt1 and neuropilin, suggesting increased responsiveness to the potent angiogenic factor VEGF. In addition, increased levels of angiopoietin-1 and ephrin B2 were observed which are involved in vascular development and stabilization. For matrix enhancing effects, considerably decreased levels (5-fold) of matrix metalloproteinases (MMPs) coupled with increased TGFbeta receptors and connective tissue growth factor (CTGF) were observed. The gene expression profile suggests that TTG alters the balance between matrix production and destruction in favor of production resulting in increased deposition of ECM in granulation tissue. In conclusion, we have identified that TTG 1) enhances fibrin-dependent wound healing response, 2) increases angiogenesis through enhanced VEGF receptors, angiopoietin-1 and ephrin B2 expression, and 3) promoted matrix deposition by simultaneously reducing MMPs and increasing CTGF and TGFbeta receptors expression.

Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1 alpha or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

Molecular imaging and therapy directed at the neovasculature in pathologies

We have discussed the impact of molecular imaging on clinical and preclinical medicine. We have presented the potential problems of delivering the effective therapeutic dose and the properties that can help contribute to the drug efficacy. The rationale for the design of new antiangiogenic agents that can be used for imaging and therapy was presented. Finally, results from imaging and targeted nanoparticle based therapies were presented. In vivo imaging of angiogenic tumors using anti-alpha(v)beta3 -targeted polymerized vesicles composed of the murine antibody LM609 attached to NPs labeled with the MR contrast agent gadolinium in the V2 carcinoma model in rabbits. MRI studies using this targeted contrast agent revealed large areas of alpha(v)beta3 integrin expression in tumor-associated vasculature that conventional MRIs failed to show. Other investigators have used microemulsions conjugated to an antibody targeted against alpha(v)beta as imaging agents. These materials also show contrast enhancement of tumor vasculature undergoing angiogenesis. Other markers, such as the PECAM-1 (CD-31), VCAM-1 (CD54) and VEGF receptor (flk-1), have been shown to be upregulated on tumor endothelium and associated with angiogenesis but have not been used in imaging studies. Furthermore, by modification of the NPs, we were able to use this imaging agent as an antiangiogenic gene delivery system. The results from these studies are very promising and are being further pursued.