Ischemia-Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts.

Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia-reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection.

Donor Heart Treatment With COMP-Ang1 Limits Ischemia-Reperfusion Injury and Rejection of Cardiac Allografts.

The major cause of death during the first year after heart transplantation is primary graft dysfunction due to preservation and ischemia-reperfusion injury (IRI). Angiopoietin-1 is a Tie2 receptor-binding paracrine growth factor with anti-inflammatory properties and indispensable roles in vascular development and stability. We used a stable variant of angiopoietin-1 (COMP-Ang1) to test whether ex vivo intracoronary treatment with a single dose of COMP-Ang1 in donor Dark Agouti rat heart subjected to 4-h cold ischemia would prevent microvascular dysfunction and inflammatory responses in the fully allogeneic recipient Wistar Furth rat. COMP-Ang1 reduced endothelial cell-cell junction disruption of the donor heart in transmission electron microscopy during 4-h cold ischemia, improved myocardial reflow, and reduced microvascular leakage and cardiomyocyte injury of transplanted allografts during IRI. Concurrently, the treatment reduced expression of danger signals, dendritic cell maturation markers, endothelial cell adhesion molecule VCAM-1 and RhoA/Rho-associated protein kinase activation and the influx of macrophages and neutrophils. Furthermore, COMP-Ang1 treatment provided sustained anti-inflammatory effects during acute rejection and prevented the development of cardiac fibrosis and allograft vasculopathy. These results suggest donor heart treatment with COMP-Ang1 having important clinical implications in the prevention of primary and subsequent long-term injury and dysfunction in cardiac allografts.

Angiopoietin-2 inhibition prevents transplant ischemia-reperfusion injury and chronic rejection in rat cardiac allografts.

Transplant ischemia-reperfusion injury (Tx-IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin-2 (Ang2) in Tx-IRI and rejection using fully MHC-mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti-Ang2 antibody before 4-h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti-Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx-IRI and, in an add-back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx-IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell-independent strategy to protect cardiac allografts.

Increased interstitial protein because of impaired lymph drainage does not induce fibrosis and inflammation in lymphedema.

OBJECTIVE: The pathophysiology of lymphedema is incompletely understood. We asked how transcapillary fluid balance parameters and lymph flow are affected in a transgenic mouse model of primary lymphedema, which due to an inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling lacks dermal lymphatics, and whether protein accumulation in the interstitium occurring in lymphedema results in inflammation. METHODS AND RESULTS: As estimated using a new optical-imaging technique, we found that this signaling defect resulted in lymph drainage in hind limb skin of K14-VEGFR-3-Ig mice that was 34% of the corresponding value in wild-type. The interstitial fluid pressure and tissue fluid volumes were significantly increased in the areas of visible swelling only, whereas the colloid osmotic pressure in plasma, and thus the colloid osmotic pressure gradient, was reduced compared to wild-type mice. An acute volume load resulted in an exaggerated interstitial fluid pressure response in transgenic mice. There was no accumulation of collagen or lipid in skin, suggesting that chronic edema presented in the K14-VEGFR-3-Ig mouse was not sufficient to induce changes in tissue composition. Proinflammatory cytokines (interleukin-2, interleukin-6, interleukin-12) in subcutaneous interstitial fluid and macrophage infiltration in skin of the paw were lower, whereas the monocyte/macrophage cell fraction in blood and spleen was higher in transgenic compared with wild-type mice. CONCLUSIONS: Our data suggest that a high interstitial protein concentration and longstanding edema is not sufficient to induce fibrosis and inflammation characteristic for the human condition and may have implications for our understanding of the pathophysiology of this condition.

Induced pluripotent stem cell clones reprogrammed via recombinant adeno-associated virus-mediated transduction contain integrated vector sequences.

Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) by ectopic expression of key transcription factors. Current methods for the generation of integration-free iPSC are limited by the low efficiency of iPSC generation and by challenges in reprogramming methodology. Recombinant adeno-associated virus (rAAV) is a potent gene delivery vehicle capable of efficient transduction of transgenic DNA into cells. rAAV stays mainly as an episome in nondividing cells, and the extent of integration is still poorly defined for various replicating cells. In this study, we aimed to induce iPSC from mouse and human fibroblasts by using rAAV vector-mediated transient delivery of reprogramming factors. We succeeded in deriving induced pluripotent stem cells from mouse but not human fibroblasts. Unexpectedly, the rAAV vector-mediated reprogramming led to frequent genomic integration of vector sequences during the reprogramming process, independent of the amount of virus used, and to persistent expression of reprogramming factors in generated iPSC clones. It thus appears that rAAV vectors are not compatible with the derivation of integration-free iPSC.

Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.

Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

Expression and prognostic value of transcription factor PROX1 in colorectal cancer.

BACKGROUND: PROX1 is a specific target of the ß-catenin/TCF pathway in the intestinal epithelium. It acts as a regulator of progression from a benign to a highly dysplastic phenotype in colorectal tumours. However, the clinical significance of PROX1 expression is not known. METHODS: We studied the prognostic value of immunohistochemical expression of PROX1 in a series of 517 patients with colorectal cancer (CRC). RESULTS: The majority of the tumour samples expressed PROX1 (91%, 471 out of 517). High PROX1 expression was associated with a poor grade of tumour differentiation (P<0.0001). In the subgroup of patients with colon cancer, high PROX1 expression was associated with unfavourable colorectal cancer-specific survival (CCSS) as compared with low PROX1 expression (CCSS 47% vs 62%; P=0.045; RR 1.47). The association between high PROX1 and poor outcome was further strengthened in female colon cancer patients (CCSS 38% vs 63%; P=0.007; RR 2.02). Nonetheless, in multivariate survival analysis PROX1 expression was not retained as an independent prognostic factor. CONCLUSION: High PROX1 expression is associated with a poor grade of tumour differentiation, and, in colon cancer patients, also with less favourable patient outcome. Our results strengthen the previous preclinical observations that PROX1 has a role in tumour progression in CRC.

Signaling angiogenesis and lymphangiogenesis.

Exciting progress has been made in elucidating the complex network of receptor-ligand interactions that regulate blood vessel growth. Understanding these control mechanisms is of interest not only because of their role in developmental biology, but because they provide potential therapeutic strategies for disease processes involving angiogenesis, such as tumor growth.

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor.

The term 'platelet-derived growth factor' (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified - the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF alpha-receptor (PDGFR-alpha) binds to all three PDGF chains, whereas the beta-receptor (PDGFR-beta) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-beta. PDGF-DD is the first known PDGFR-beta-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Clinical applications of angiogenic growth factors and their inhibitors.

Promoting the formation of new collateral vessels in ischemic tissues using angiogenic growth factors (therapeutic angiogenesis) is a an exciting frontier of cardiovascular medicine. Conversely, inhibition of the action of key regulators of angiogenesis, such as VEGF, constitutes a promising approach for the treatment of solid tumors and intraocular neovascular syndromes. These concepts are being tested now in clinical trials.

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.

Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

The lymphatic vasculature transports extravasated tissue fluid, macromolecules and cells back into the blood circulation. Recent reports have focused on the molecular mechanisms regulating the lymphatic vessels. Vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to stimulate lymphangiogenesis and their receptor, VEGFR-3, has been linked to human hereditary lymphedema. Here we show that a soluble form of VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signaling, and when expressed in the skin of transgenic mice, it inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels, though the blood vasculature remains normal. Transgenic mice develop a lymphedema-like phenotype characterized by swelling of feet, edema and dermal fibrosis. They survive the neonatal period in spite of a virtually complete lack of lymphatic vessels in several tissues, and later show regeneration of the lymphatic vasculature, indicating that induction of lymphatic regeneration may also be possible in humans.

Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

Fibronectin is produced by human macrophages.

Monocyte-enriched cultures were prepared from human blood mononuclear leukocytes by adherence to growth substratum. Synthesis and secretion of fibronectin was detected in these cultures concomitantly with morphological differentiation, starting on day 3--5. Production of fibronectin by macrophages was documented by metabolic labeling followed by immunoprecipitation and gel electrophoresis, radioimmunoassay specific for human fibronectin, and by indirect immunofluorescence. Fibronectin was detected mainly intracellularly but was also detected pericellularly only in minute amounts. No production of collagenous proteins was seen in these cultures. Macrophage fibronectin might act in vivo as a nonspecific opsonin and promote cell adhesion during macrophage migration in tissues.

The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells.

The growth factor receptors expressed on endothelial cells are of special interest because of their potential to program endothelial cell growth and differentiation during development and neovascularization in various pathological states, such as wound healing and angiogenesis associated with tumorigenesis. Vascular endothelial growth factor ([VEGF] also known as vascular permeability factor) is a potent mitogen and permeability factor, which has been suggested to play a role in embryonic and tumor angiogenesis. The newly cloned FLT4 receptor tyrosine kinase gene encodes a protein related to the VEGF receptors FLT1 and KDR/FLK-1. We have here studied the expression of FLT4 and the other two members of this receptor family in human fetal tissues by Northern and in situ hybridization. These results were also compared with the sites of expression of VEGF and the related placenta growth factor (PlGF). Our results reveal FLT4 mRNA expression in vascular endothelial cells in developing vessels of several organs. A comparison of FLT4, FLT1 and KDR/FLK-1 receptor mRNA signals shows overlapping, but distinct expression patterns in the tissues studied. Certain endothelia lack one or two of the three receptor mRNAs. These data suggest that the receptor tyrosine kinases encoded by the FLT gene family may have distinct functions in the regulation of the growth/differentiation of blood vessels.

Recessive primary congenital lymphoedema caused by a VEGFR3 mutation.

BACKGROUND: Heterozygous mutations in VEGFR3 have been identified in some familial cases with dominantly inherited primary congenital lymphoedema, known as Nonne-Milroy disease. Recessive cases of primary lymphoedema with a genetic cause are not known, except for two families with syndromic hypotrichosis-lymphoedema-telangiectasia, with a SOX18 mutation. METHODS AND RESULTS: In this study, we present the first case of isolated primary congenital lymphoedema with recessive inheritance, caused by a homozygous mutation in VEGFR3. The novel mutation is a transition from alanine-to-threonine in amino acid 855, located in the ATP binding domain of the VEGFR3 receptor. Assessment of receptor function showed impaired ligand induced internalisation and ERK1/2 activity. Moreover, receptor phosphorylation was reduced, although less so than for a kinase-dead VEGFR3 mutation, which causes Nonne-Milroy disease. CONCLUSION: A hypomorphic VEGFR3 mutation, with moderate effect on receptor function, in a homozygous state can result in insufficient lymphatic functioning. Thus, in addition to Nonne-Milroy disease with dominant inheritance, VEGFR3 alterations can cause isolated recessive primary congenital lymphoedema. These data expand our understanding of the aetiology of congenital lymphoedema and suggest that large scale screening of VEGFR3 in all primary lymphoedema patients is necessary.

c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts display more decondensed nucleosomal organization than normal fibroblasts.

We have compared the nucleosomal organization of c-Ha-rasVal 12 oncogene-transformed NIH-3T3 fibroblasts with that of normal fibroblasts by using micrococcal nuclease (MNase) as a probe for the chromatin structure. The bulk chromatin from asynchronously and exponentially growing ras-transformed cells was much more sensitive to MNase digestion than chromatin from the normal cells. Southern hybridization analyses of the MNase digests with probes specific for the ornithine decarboxylase (odc) and c-myc genes showed that the coding and/or 3' end regions of these growth-inducible genes carry a nucleosomal organization both in ras-transformed and normal cells. Studies with cells synchronized by serum starvation showed that in both cell lines the nucleosomal organization of chromatin is relatively condensed at the quiescent state, becomes highly decondensed during the late G1 phase of the cell cycle, and starts again to condense during the S phase. However, in ras-transformed cells the decondensation state stayed much longer than in normal cells. Moreover, irrespective of the phase of the cell cycle the bulk chromatin as well as that of the odc and c-myc genes was more sensitive to MNase digestion in the ras-transformed cell than in the normal fibroblast. Decondensation of the chromatin was also observed in the normal c-Ha-ras protooncogene-transfected cells, but to a lesser extent than in the mutant ras-transformed cells. Whether the increased degree of chromatin decondensation plays a regulatory role in the increased expression of many growth-related genes in the ras-transformed cells remains an interesting object of further study.

Activation of the neu tyrosine kinase induces the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase.

We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF-R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP-1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation.

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells.

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.