Showing papers by "Kari Alitalo published in 2006"

Signaling and Functions of Angiopoietin-1 in Vascular Protection

Abstract: Angiopoietin-1 (Ang1) has powerful vascular protective effects: suppressing plasma leakage, inhibiting vascular inflammation, and preventing endothelial death. Preclinical studies indicate that Ang1 may be therapeutically useful in a number of situations, including treatment of edema, endotoxemia, and transplant arteriosclerosis. However, the ligand has also been implicated in vessel remodeling, induction of angiogenesis and pulmonary hypertension, indicating that strategies to minimize any deleterious effects while optimizing vessel protection are likely to be needed. This review surveys the published data on vascular protective effects of Ang1 and highlights the therapeutic potential of this ligand, as well as possible limitations to its use. We also consider the data on Ang1 receptors and speculate on how to maximize therapeutic benefit by targeting the Tie receptors.

Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.

Abstract: Lymphatic vascular development is regulated by vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by its ligands VEGF-C and VEGF-D. Neuropilin-2 (NP2), known to be involved in neuronal development, has also been implicated to play a role in lymphangiogenesis. We aimed to elucidate the mechanism by which NP2 is involved in lymphatic endothelial cell signaling. By in vitro binding studies we found that both VEGF-C and VEGF-D interact with NP2, VEGF-C in a heparin-independent and VEGF-D in a heparin-dependent manner. We also mapped the domains of VEGF-C and NP2 required for their binding. The functional importance of the interaction of NP2 with the lymphangiogenic growth factors was demonstrated by cointernalization of NP2 along with VEGFR-3 in endocytic vesicles of lymphatic endothelial cells upon stimulation with VEGF-C or VEGF-D. NP2 also interacted with VEGFR-3 in coprecipitation studies. Our results show that NP2 is directly involved in an active signaling complex with the key regulators of lymphangiogenesis and thus suggest a mechanism by which NP2 functions in the development of the lymphatic vasculature.

Vascular Endothelial Growth Factor-C Accelerates Diabetic Wound Healing

Abstract: Diabetes impairs numerous aspects of tissue repair. Failure of wound angiogenesis is known to delay diabetic wound healing, whereas the importance of lymphangiogenesis for wound healing is unclear. We have examined whether overexpression of vascular endothelial growth factor (VEGF)-C via an adenoviral vector could improve the healing of full-thickness punch biopsy wounds in genetically diabetic (db/db) mice. We found that VEGF-C enhanced angiogenesis and lymphangiogenesis in the wound and significantly accelerated wound healing in comparison to the control wounds. VEGF-C also recruited inflammatory cells, some of which expressed VEGFR-3. On the other hand, when the function of endogenous VEGF-C/VEGF-D was blocked with a specific inhibitor, wound closure was delayed even further. These results suggest a function for VEGF-C in wound healing and demonstrate the therapeutic potential of VEGF-C in the treatment of diabetic wounds.

Large-scale identification of genes implicated in kidney glomerulus development and function.

Abstract: To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large-scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non-glomerular kidney tissue, FACS-sorted podocytes and brain capillaries, which identified over 300 glomerular cell-enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus-enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain.

Abstract: Vascular endothelial growth factor C (VEGF-C) was first identified as a regulator of the vascular system, where it is required for the development of lymphatic vessels. Here we report actions of VEGF-C in the central nervous system. We detected the expression of the VEGF-C receptor VEGFR-3 in neural progenitor cells in Xenopus laevis and mouse embryos. In Xenopus tadpole VEGF-C knockdowns and in mice lacking Vegfc, the proliferation of neural progenitors expressing VEGFR-3 was severely reduced, in the absence of intracerebral blood vessel defects. In addition, Vegfc-deficient mouse embryos showed a selective loss of oligodendrocyte precursor cells (OPCs) in the embryonic optic nerve. In vitro, VEGF-C stimulated the proliferation of OPCs expressing VEGFR-3 and nestin-positive ventricular neural cells. VEGF-C thus has a new, evolutionary conserved function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3.

Lymphangiogenesis and cancer metastasis.

Abstract: Proc Amer Assoc Cancer Res, Volume 47, 2006 SY11-01 The induction of angiogenesis and neovascularization is an important mechanism by which tumors promote their continued growth and subsequent metastasis The lymphatic vascular system offers another particularly apt conduit for the spread of tumor cells, as this network of vessels functions naturally as an exit route for immune cells from tissues Indeed, the propensity for lymphatic metastasis is a common feature of most solid tumors In contrast to tumors that are composed of heterogenous and adaptive malignant cells, which easily evolve to become resistant to treatment, blood and lymphatic endothelial cells represent normal diploid cells and they should only grow in response to growth factors secreted by tumor cells Recent successes in the treatment of colorectal cancer patients with monoclonal neutralizing antibodies to vascular endothelial growth factor (VEGF) have demonstrated the potential of highly specific molecular therapies targeting endothelial cells and the angiogenic process Similar approaches could eventually be used to target lymphatic vessels for the development of anti-metastatic therapies Despite the importance of the lymphatic vasculature very little was known, until recently, about the mechanisms of lymph vessel growth and development Lymphatic vessels were first described in the 17th century and their development described a century ago However it was only 10 years ago that any growth factors and molecular markers specific to lymph vessels were discovered, sparking intense activity in the field The lymphatic vasculature is essential for the maintenance of interstitial fluid balance, for immune defence and for the uptake of dietary fat Lymphatic aplasia, hypoplasia or obstructive, obliterative or surgical hindrance of lymph drainage causes stagnation of proteins and associated water in the interstitium, and leads to lymphedema, a disfiguring and disabling swelling of the extremities Dissemination of tumor cells via lymphatic vessels to distant organs is a leading cause of death in cancer patients, and a major obstacle in the design of effective therapies Even though metastasis via the lymph is common in cancers, little attention has been paid to the lymphatic vasculature in tumors and the mechanisms regulating its development and growth The situation has changed during the past few years, mainly because of progress in the identification of regulatory molecules and markers specific for the lymphatic endothelial cells Lymphangiogenesis accompanies angiogenesis in both embryogenesis and disease, yet the morphologic and functional differences between lymphatic and blood capillaries are striking The basis for these differences may lie in the genes expressed by lymphatic and blood endothelial cells Lymphatic endothelial cell differentiation commitment and growth during the embryonic development are dependent on the activities of the homeodomain transcription factor Prox-1 and VEGF-C, respectively Comparison of the transcriptional profiles of blood vessel and lymphatic vessel endothelial cells on a genome-wide scale has opened the possibility to systematically analyze differences between these two cell lineages The transcripts expressed by lymphatic endothelial cells and blood vascular endothelial cells are modified soon after their isolation from tissues and approximately 2% of transcribed genes are differentially expressed between these two cell types For example, blood vascular endothelial cells express significantly higher levels of pro-inflammatory cytokines, chemokines and chemokine receptors, have higher levels of β-catenin and display increased numbers of actin stress fibers The genetic programs that determine the complexity of lymphatic capillaries and the larger collecting lymphatic vessels and make them distinct from blood vessels involve several other newly described signal transduction pathways For example, the FOXC2 transcription factor has been implicated in the formation of the lymph vessels and their valves As VEGF-C and VEGF-D induce adult lymphangiogenesis, associated with inflammation, wound healing and tumor metastasis, administration of lymphatic growth factors or their antagonists could provide the opportunity to target lymphatic vessels in human disease Five members of the human VEGF family interact with the VEGF receptors (VEGFRs) to relay the intracellular signals responsible for the induction of growth, migration and survival of endothelial cells In addition, the VEGFs interact with neuropilin coreceptors In the lymphatic vessels VEGF-C and VEGF-D interact with VEGFR-3 and neuropilin-2 Neuropilin-2 also serves as a plexin co-receptor for type III semaphorins in the nervous system Although all three VEGFRs are required for the development of blood vessels in embryos, VEGFR-3 expression becomes largely confined to the lymphatic endothelium in adults Additional, less explored signal transduction pathways include the interaction of VEGF-C with integrin α9, and VEGFR-3 with integrin β1 and human Kaposi sarcoma herpesvirus-8 envelope protein gB After proteolytic cleavage of the N- and C-terminal propeptides of VEGF-C and VEGF-D these proteins acquire the ability to activate VEGFR-2, also present in the lymphatic endothelium Correspondingly, the processed forms of VEGF-C and VEGF-D are able to induce angiogenesis via the VEGFR-2 on blood vessels Although studies have clearly shown that lymphangiogenesis is induced by VEGF-C and VEGF-D via VEGFR-3 activation, recent findings also suggest that VEGF, angiopoietin-1, and fibroblast, hepatocyte and insulin-like growth factors may also mediate lymphangiogenesis directly or indirectly In most cancers, the metastatic spread of tumor cells to distant organs results in patient mortality Tumor metastasis occurs to lymph nodes and distant organs via lymph or blood vessels The patterns of metastasis vary from tumor to tumor, but regional lymph nodes are often the first site of metastasis in carcinomas Metastatic nodes are important in the staging, treatment and follow-up of many solid tumors Regional lymph node metastasis is often the most important prognostic factor for patients with malignant tumors of epithelial origin The sentinel lymph node is the first regional lymph node to be invaded by tumor cells; further metastasis may then occur to other nodes and to the systemic circulation The process of metastasis is complex and involves changes in the expression of many genes In most tumors, it is not known if metastasis via the lymph selects for cells with high affinity for a distant destination, or if the appearance of lymphatic metastasis just indicates that the tumor has entered a metastatic phase in general, including organ metastasis Growth factor stimulation of lymphatic vessels appears to enhance lymphatic metastasis, but many questions about the mechanisms of lymphatic dissemination remain unanswered VEGF-C and VEGF-D expression correlate with vascular invasion, lymphatic vessel and lymph node involvement, distant metastasis, and, in some instances, poor clinical outcomes Also studies of various tumor models have shown that VEGF-C and VEGF-D overexpression can enhance lymphatic metastasis Furthermore, a soluble VEGFR-3 fusion protein (“VEGF-C/D Trap”) inhibited lymphatic metastasis in several tumor models In some models lymphatic, but not lung metastases were blocked with a VEGF-C/D trap while in others the treatment inhibited both lymph node and lung metastases Although these experiments provide strong support for the involvement of VEGF-C, VEGF-D, and their receptor VEGFR-3 in the lymphatic spread of malignancy, the underlying mechanisms have only recently been addressed Lymph vessel proliferation seen in tumor models overexpressing the lymphangiogenic factors may not be a prominent feature in several human cancers, and may in fact not be needed for enhanced metastasis in most solid tumors While intratumoral lymphatic vessels have been detected in some solid tumors, such as melanomas and head and neck carcinomas, at least in experimental tumors they may not be completely functional, because of their collapse in conditions of high intratumoral pressure On the other hand, the pressure gradient and lymph vessels at the tumor margin may be more important in spreading tumor cells through the process of vessel sprouting stimulated by tumor-secreted VEGF-C or VEGF-D In this process the endothelial cells send long filopodia towards the growth factor producing tumor cells and then form tumor-directed vessel sprouts, where the vessel lumen opens up and may allow facilitated access of tumor cells to the lumen In some cases the lymphatic endothelium could also actively participate in metastasis formation by secreting chemokines that have receptors in tumor cells Furthermore, the collecting lymphatic vessels draining fluid from the tumor area are stimulated to dilate by intraluminal VEGF-C via the process of endothelial proliferation in the vessel wall Clumps of metastatic tumor cells could then undergo an easier transit in lymph flowing in the dilated hyperplastic vessels The VEGF-C/D trap inhibited sprouting and vessel dilation, and seemed to restore the integrity of the vessel wall Similarly, blocking monoclonal antibodies that target VEGF-C, VEGF-D or their receptor(s) and small molecules that inhibit the tyrosine kinase catalytic domain of these receptors could be used for the inhibition of experimental tumor metastasis Further work should soon tell if these same molecules inhibit further systemic metastasis or angiogenesis in tumor models

Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation. Commentary

Abstract: Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (Ad-VEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangio-genesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.

Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation.

Abstract: Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangiogenesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.

Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis

Abstract: Bmx/Etk non-receptor tyrosine protein kinase has been implicated in endothelial cell migration and tube formation in vitro. However, the role of Bmx in vivo is not known. Bmx is highly induced in the vasculature of ischemic hind limbs. We used both mice with a genetic deletion of Bmx (Bmx-KO mice) and transgenic mice expressing a constitutively active form of Bmx under the endothelial Tie-2 enhancer/promoter (Bmx-SK-Tg mice) to study the role of Bmx in ischemia-mediated arteriogenesis/angiogenesis. In response to ischemia, Bmx-KO mice had markedly reduced, whereas Bmx-SK-Tg mice had enhanced, clinical recovery, limb perfusion, and ischemic reserve capacity when compared with nontransgenic control mice. The functional outcomes in these mice were correlated with ischemia-initiated arteriogenesis, capillary formation, and vessel maturation as well as Bmx-dependent expression/activation of TNF receptor 2– and VEGFR2-mediated (TNFR2/VEGFR2-mediated) angiogenic signaling in both hind limb and bone marrow. More importantly, results of bone marrow transplantation studies showed that Bmx in bone marrow–derived cells plays a critical role in the early phase of ischemic tissue remodeling. Our study provides the first demonstration to our knowledge that Bmx in endothelium and bone marrow plays a critical role in arteriogenesis/angiogenesis in vivo and suggests that Bmx may be a novel target for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.

Early Lymph Vessel Development From Embryonic Stem Cells

Abstract: Objective— The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of murine embryonic stem cells. Methods and Results— Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics. Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of ...

Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

Abstract: Objective— Vascular endothelial growth factor (VEGF) plays critical roles in the regulation of angiogenesis and lymphangiogenesis. However, tissue edema, hemorrhage, and inflammation occur when VEGF-A is used for angiogenic therapy. To design a novel angiogenic factor without severe side effects, we examined the biological function of chimeric VEGF-E NZ7 /placental growth factor (PlGF), which is composed of Orf-Virus NZ7 -derived VEGF-E NZ7 and human PlGF1, in a transgenic (Tg) mouse model. Methods and Results— A strong angiogenic response was observed in both VEGF-E NZ7 /PlGF and VEGF-A 165 Tg mice. Notably, the vascular leakage of VEGF-E NZ7 /PlGF-induced blood vessels was 4-fold lower than that of VEGF-A 165 –induced blood vessels. Furthermore, the monocyte/macrophage recruitment in the skin of VEGF-E NZ7 /PlGF Tg mice was &8-fold decreased compared with that of VEGF-A 165 Tg mice. In addition, the lymphatic vessels in VEGF-E NZ7 /PlGF Tg mice were structurally normal, whereas they were markedly dilated in VEGF-A 165 Tg mice, possibly because of the high vascular leakage. Receptor binding assay demonstrated that VEGF-E NZ7 /PlGF was the ligand only activating VEGF receptor (VEGFR)-2. Conclusion— These results indicated that neither the hyperpermeability in response to simultaneous stimulation of VEGFR-1 and VEGFR-2 nor VEGFR-1–mediated severe inflammation was associated with VEGF-E NZ7 /PlGF-induced angiogenesis. The unique receptor binding property may shed light on VEGF-E NZ7 /PlGF as a novel candidate for therapeutic angiogenesis.

Ocular versus extraocular neovascularization: Mirror images or vague resemblances

Abstract: The First ARVO/Pfizer Ophthalmics Institute meeting was held Friday and Saturday, April 29 and 30, 2005, in Fort Lauderdale, Florida, in conjunction with the 2005 ARVO Annual Meeting. The Institute meeting was funded by The ARVO Foundation for Eye Research through a grant from Pfizer Ophthalmics. The goal of the Institute is to develop strategies to improve research and clinical care in areas of ophthalmology related to preventable vision loss and blindness. The specific topic and organizer were selected by an independent selection committee which reviewed proposals received through an open call for proposals related to angiogenesis, neovascularization, and vasoproliferation. The proposal organizer solicited an international, interdisciplinary panel of expert speakers that comprised the First ARVO/Pfizer Ophthalmics Institute Working Group. One outcome of the Institute is publication of the research presented and discussions that followed to share possible new opportunities for research with the vision community and its partners in other disciplines.

Transcapillary fluid balance consequences of missing initial lymphatics studied in a mouse model of primary lymphoedema

Abstract: To investigate the phenotypic consequences of a deranged lymphangiogenesis in relation to tissue fluid accumulation and the possible role of inflammation in the pathogenesis of lymphoedema, we measured determinants of transcapillary fluid filtration and inflammatory mediators in the interstitial fluid in genetically engineered Chy mice, a model for primary congenital lymphoedema (Milroy's disease). Although initial lymphatics were not present in dermis in any of the areas studied (fore paw, hind paw, thigh and back skin) interstitial fluid pressure (Pif), measured with micropipettes, and tissue fluid volumes were significantly increased only in the areas with visible swelling – the fore and hind paw, whereas interstitial colloid osmotic pressure (COPif) was increased in all the skin areas examined. A volume load of 15% of body weight resulted in a more pronounced increase in Pif as well as a four-fold increase in interstitial fluid volume in Chy relative to wild-type (wt) mice, showing the quantitative importance of lymphatics for fluid homeostasis during acute perturbations. A similar level of proinflammatory markers in interstitial fluid in early established lymphoedema (3–4 months) in Chy and wt suggests that inflammation does not have a major pathogenetic role for the development of lymphoedema, whereas a reduced level of the immunomodulatory cytokine interleukin (IL)-4 may result in a reduced immunological defence ability and thus lead to the increase in inflammatory cytokines IL-2 and IL-6 observed at a later stage (11–13 months). Our data suggest that primary lymphoedema results in a high interstitial fluid protein concentration that does not induce an interstitial inflammatory reaction per se, and furthermore shows the paramount importance of the initial lymphatics in tissue fluid homeostasis, especially during perturbations of transcapillary fluid balance.

Vascular Endothelial Growth Factor C–induced Collateral Formation in a Model of Myocardial Ischemia

Abstract: Background Besides being a known lymphangiogenic activator, vascular endothelial growth factor (VEGF)-C may express angiogenic potential by proteolytic cleavage and activation of endothelial cells. We assessed myocardial collateral formation and functional changes after adenovirus-mediated VEGF-C gene transfer in an ischemic porcine model. Methods Fifteen Landrace piglets underwent Ameroid-induced gradual occlusion of the left circumflex artery (LCx) and consequent progressive myocardial ischemia. Three weeks after Ameroid placement, the animals underwent gated 99m Tc SPECT during rest and stress, in vivo angiography and 18 FDG PET. Pigs were randomized to intramyocardial injections of adenoviruses encoding vascular endothelial growth factor (VEGF-C; n = 7) or control beta-galactosidase (LacZ; n = 5). Four weeks later, the examinations were repeated and histology was analyzed. Results Angiography showed significant progression of LCx stenosis in both groups during the treatment period. Left ventricular wall thickening (LVWT) at the LCx area in gated 99m Tc SPECT remained unchanged in the VEGF-C group, indicating that VEGF-C prevented progression of myocardial ischemia, whereas LVWT deteriorated in the LacZ group ( p = 0.042). Semi-quantitative assessment of 18 FDG PET suggests more reduction in ischemia in the adVEGF-C group than in controls ( p = 0.052). Angiography showed significant clustering of collaterals in the adVEGF-C gene transfer area compared that in LacZ ( p = 0.004). von Willebrand factor staining revealed a significantly ( p = 0.03) greater number of microvessels in the adVEGF-C–treated myocardium. Conclusions This appears to be the first large-animal study in which, during progressive ischemia, functional and metabolic benefits of intramyocardial VEGF-C gene transfer were apparent. VEGF-C–induced collateral formation occurred at the site of gene transfer. The angiogenic potency of VEGF-C deserves further study as a therapeutic option.

Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy.

Abstract: Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.

Pulmonary vascular endothelial growth factor-C in development and lung injury in preterm infants.

Abstract: Rationale: In mice, vascular endothelial growth factor-C (VEGF-C) plays an important role in development of the lymphatic system and in pathogenesis of pulmonary inflammation. Its role in development of the lymphatic system in human lung and in lung injury in newborns remains unclear.Objectives: We studied the role of VEGF-C in developing human lung, and in acute and chronic lung injury in preterm infants.Methods: Included in the immunohistochemistry study were 10 fetuses, 15 control neonates without primary lung disease, 15 preterm infants with respiratory distress syndrome, and 8 infants with bronchopulmonary dysplasia. Tracheal aspirate fluid samples of intubated very-low-birth-weight infants during Postnatal Weeks 1–5 were analyzed with ELISA.Results: Bronchiolar staining for VEGF-C was observed in all 48 samples. Alveolar epithelial staining was seen in most fetuses (8/10). In addition, staining was observed in alveolar macrophages in bronchopulmonary dysplasia (4/8), and late respiratory distress sy...

The effect of platelet-derived growth factor ligands in rat cardiac allograft vasculopathy and fibrosis.

Abstract: Background In chronic rejection, parenchymal fibrosis and cardiac allograft vasculopathy (CAV) characterized by neointimal growth are the leading causes of graft loss for heart transplant recipients. During alloimmune responses a variety of cytokines, adhesion proteins, and growth factors, such as platelet-derived growth factor (PDGF), are up-regulated. The PDGF family (AA, AB, BB, CC, DD), which acts mainly on connective tissue cells, is considered to be a potent mitogenic and chemotactic factor for fibroblasts and vascular smooth muscle cells. In this study, we evaluated the effects of PDGF ligands in chronic rejection. Methods Heterotopic heart transplantations were performed between fully major histocompatability complex–mismatched Dark Agouti to Wistar Furth rats receiving cyclosporine immunosuppression. Allograft coronary arteries were perfused with a recombinant adeno-associated virus (AAV) encoding enhanced green fluorescence protein (EGFP) as a control gene or PDGF-A, -B, -C, -D. Allografts were harvested at 100 days for morphometric analysis of CAV and fibrosis. Results AAV-mediated transgene expression was detected by EGFP immunoreactivity across the graft section (at 100 days) in AAV EGFP-perfused allografts. AAV PDGF-A, -C, and -D perfusion resulted in accelerated CAV and fibrosis. In contrast, AAV PDGF-B perfusion did not induce arteriosclerotic changes or fibrosis in cardiac allografts. Conclusions AAV PDGF-A, -C, and -D overexpression accelerated the development of chronic rejection, whereas PDGF-B did not. Our results suggested that more targeted therapy with monoclonal antibodies blocking the active sites of PDGF-A, -C, and -D may produce beneficial effects on heart transplant survival.

Short and Long-Term Effects of hVEGF-A165 in Cre-Activated Transgenic Mice

Abstract: We have generated a transgenic mouse where hVEGF-A165 expression has been silenced with loxP-STOP fragment, and we used this model to study the effects of hVEGF-A165 over-expression in mice after systemic adenovirus mediated Cre-gene transfer. Unlike previous conventional transgenic models, this model leads to the expression of hVEGF-A165 in only a low number of cells in the target tissues in adult mice. Levels of hVEGF-A165 expression were moderate and morphological changes were found mainly in the liver, showing typical signs of active angiogenesis. Most mice were healthy without any major consequences up to 18 months after the activation of hVEGF-A165 expression. However, one mouse with a high plasma hVEGF-A165 level died spontaneously because of bleeding into abdominal cavity and having liver hemangioma, haemorrhagic paratubarian cystic lesions and spleen peliosis. Also, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in control mice. We conclude that long-term uncontrolled hVEGF-A165 expression in only a limited number of target cells in adult mice can be associated with pathological changes, including possible formation of malignant tumors and uncontrolled bleeding in target tissues. These findings have implications for the design of long-term clinical trials using hVEGF-A165 gene and protein.

Modified VEGF-A with improved angiogenic properties

Abstract: The present invention is directed to methods and compositions for making and using chimeric polypeptides that comprise a VEGFR-2 ligand. The chimeric molecules of the present invention retain VEGFR-2 binding activity and an enhanced angiogenic activity as compared to native VEGF-A.

Moderate antiangiogenic activity by local, transgenic expression of endostatin in Rip1Tag2 transgenic mice

Abstract: Many previous reports have demon- strated that systemic administration of endostatin (ES), a proteolytic cleavage product of collagen type XVIII and an endogenous angiogenesis inhib- itor, represses tumor angiogenesis in different pre- clinical tumor models with varying efficacy. For example, systemic delivery of recombinant ES to rat insulin promoter 1 (Rip1)T-antigen 2 (Tag2)- transgenic mice, a mouse model of pancreatic -cell carcinogenesis, has repressed tumor angio- genesis efficiently and with it, tumor growth. Here, we report that the transgenic expression of ES in Rip1ES-transgenic mice only interferes moder- ately with tumor growth in Rip1Tag2;Rip1ES dou- ble-transgenic mice. Tumor incidence is not re- duced by the local expression of ES, and tumor outgrowth and progression to tumor malignancy are only retarded slightly. A significant effect of local ES expression on tumor angiogenesis is only apparent during the early stages of tumor develop- ment, where less angiogenic hyperplastic lesions are observed. Although efficiently produced and secreted by transgenic cells, locally expressed ES appears to be sequestered in the microenviron- ment, and its systemic levels are not increased. The results indicate that the antiangiogenic functions of ES critically depend on the mode of delivery and the site of expression: although its systemic appli- cation represses tumor angiogenesis and tumor growth efficiently, locally expressed ES appears to be less effective, and hence, additional mechanisms of solubilization or activation of latent ES seem to be required. These results have important implica- tions about the modes of delivery used in antian- giogenic, therapeutic strategies, which are based on the antiangiogenic activities of ES. J. Leukoc. Biol. 80: 669-676; 2006.

Modified vegf and pdgf with improved angiogenic properties

Abstract: The present invention is directed to methods and compositions for making and using chimeric polypeptides that comprise a VEGFR-2 ligand. The chimeric molecules of the present invention retain VEGFR-2 binding activity and an enhanced angiogenic activity as compared to native VEGF-A.