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Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies., (© 2013 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Background: Hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxia, affects a wide range of adaptive cell functions. We examined the kinetics of endogenous HIF-1alpha during acute and chronic rejection, and the effect of exogenous HIF-1alpha in chronically rejecting rat cardiac allografts., Methods: Heterotopic cardiac transplantations were performed between major MHC-mismatched Dark Agouti and Wistar-Furth rats. Cyclosporine A (CsA) was used to prevent acute rejection in the chronic rejection model. The effect of HIF-1alpha overexpression was investigated by adeno-assocated virus 2 (AAV2)-mediated gene transfer of a constitutively stabilized form of mouse HIF-1alpha (AAV-HIF-1alpha). The analysis of allografts was based on histology, immunohistochemistry and quantitative reverse transcript-polymerase chain reaction (RT-PCR)., Results: Acute and chronic rejection significantly induced HIF-1alpha mRNA in rat cardiac allografts when compared with syngeneic controls. Immunohistochemistry localized significantly increased HIF-1alpha immunoreactivity to vascular smooth muscle cells, vascular endothelial cells, post-capillary venules and graft-infiltrating mononuclear inflammatory cells of the allograft, whereas expression in cardiomyocytes remained unchanged. Regression analysis revealed a linear correlation between the progression of cardiac allograft vasculopathy (CAV) and HIF-1alpha immunoreactivity in post-capillary venules and graft-infiltrating mononuclear inflammatory cells in chronically rejecting rat cardiac allografts. AAV-HIF-1alpha enhanced cardiomyocyte HIF-1alpha production and significantly reduced cardiomyocyte apoptosis and the development of CAV in chronically rejecting rat cardiac allografts., Conclusions: We found that acute and chronic rejection increased HIF-1alpha mRNA and protein levels in rat cardiac allografts. On the other hand, cardiomyocyte-targeted HIF-1alpha gene transfer inhibited cardiomyocyte apoptosis and the development of CAV, suggesting a novel therapeutic strategy for HIF-1alpha in cardiac allografts., (Copyright 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown that the maintenance of post-mitotic state is critical for the life-long survival of the inner ear mechanosensory cells, the hair cells. A general concept is that differentiated, post-mitotic cells rapidly die following cell cycle re-entry. Here we have compared the response of postnatal cochlear (auditory) and utricular (balance) hair cells to forced cell cycle reactivation and p53 up-regulation. Forced S-phase entry was triggered through the human papillomavirus-16 E7 oncogene misexpression in explant cultures. It induced DNA damage and p53 induction in cochlear outer hair cells and these cells were rapidly lost, before entry into mitosis. The death was attenuated by p53 inactivation. In contrast, despite DNA damage and p53 induction, utricular hair cells showed longer term survival and a proportion of them progressed into mitosis. Consistently, pharmacological elevation of p53 levels by nutlin-3a led to a death-prone phenotype of cochlear outer hair cells, while other hair cell populations were death-resistant. These data have important clinical implications as they show the importance of p53 in sensory cells that are essential in hearing function.

Objective: Chronic rejection is the main reason for the poor long-term survival of heart transplant recipients and is characterized by cardiac allograft inflammation, fibrosis, and arteriosclerosis. We examined the specific roles of different platelet-derived growth factor (PDGF) ligands (A-D)--potent mesenchymal cell mitogens--in rat cardiac allografts., Methods and Results: PDGFR-alpha mRNA was upregulated in acutely-rejecting, and PDGF-A and PDGF-C mRNA in chronically-rejecting cardiac centhatn allografts. In acute rejection, PDGFR-alpha immunoreactivity increased in the media of arteries. In chronically-rejecting allografts, immunoreactivity of all PDGF ligands and receptors--except that of PDGF-B ligand--was found in the intima of arteries, and the expression of PDGF-A and PDGF-C was seen in cardiomyocytes. Intracoronary adeno-associated virus-2 (AAV2)-mediated PDGF-A and -D gene transfer enhanced cardiac allograft inflammation. AAV2-PDGF-A, AAV2-PDGF-C, and AAV2-PDGF-D significantly upregulated profibrotic TGF-beta1 mRNA and accelerated cardiac fibrosis and arteriosclerosis. In contrast, AAV2-PDGF-B did not aggravate chronic rejection., Conclusions: We found that alloimmune response induces PDGF-A, PDGF-C, and PDGF-D expression in the graft vasculature. PDGF-A, PDGF-C, and PDGF-D mediated profibrotic and proarteriosclerotic effects in transplanted hearts involving the TGF-beta1 pathway. Inhibition of signaling of all PDGF-ligands except that of PDGF-B may thus be needed to inhibit chronic rejection in cardiac allografts.

Vascular endothelial growth factor (VEGF)-C and VEGF-D are composed of the receptor-binding VEGF homology domain and a carboxy-terminal silk homology domain that requires proteolytic cleavage for growth factor activation. Here, we explored whether the C-terminal heparin-binding domain of the VEGF(165) or VEGF(189) isoform also containing neuropilin-binding sequences could substitute for the silk homology domain of VEGF-C. Such VEGF-C/VEGF-heparin-binding domain chimeras were produced and shown to activate VEGF-C receptors, and, when expressed in tissues via adenovirus or adeno-associated virus vectors, stimulated lymphangiogenesis in vivo. However, both chimeras induced a distinctly different pattern of lymphatic vessels when compared with VEGF-C. Whereas VEGF-C-induced vessels were initially a dense network of small diameter vessels, the lymphatic vessels induced by the chimeric growth factors tended to form directly along tissue borders, along basement membranes that are rich in heparan sulfate. For example, in skeletal muscle, the chimeras induced formation of lumenized lymphatic vessels more efficiently than wild-type VEGF-C. We conclude that the matrix-binding domain of VEGF can target VEGF-C activity to heparin-rich basement membrane structures. These properties may prove useful for tissue engineering and attempts to regenerate lymphatic vessels in lymphedema patients.

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.

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia-inducible factor-1alpha (HIF-1alpha). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF-1alpha markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF-1alpha overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF-1alpha treated muscles was significantly enhanced when compared to the controls and not further improved by co-expression of the arteriogenic growth factors angiopoietin-1 or platelet-derived growth factor-B. Our results show that AAV-mediated transduction of a stabilized form of HIF-1alpha can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF-1alpha should thus offer a potent alternative for pro-angiogenic gene therapy.

Angiopoietin 1 (Ang1), a ligand for the receptor tyrosine kinase Tie2, regulates the formation and stabilization of the blood vessel network during embryogenesis. In adults, Ang1 is associated with blood vessel stabilization and recruitment of perivascular cells, whereas Ang2 acts to counter these actions. Recent results from gene-targeted mice have shown that Ang2 is also essential for the proper patterning of lymphatic vessels and that Ang1 can be substituted for this function. In order to characterize the effects of the angiopoietins on lymphatic vessels, we employed viral vectors for overexpression of Ang1 in adult mouse tissues. We found that Ang1 activated lymphatic vessel endothelial proliferation, vessel enlargement, and generation of long endothelial cell filopodia that eventually fused, leading to new sprouts and vessel development. Cutaneous lymphatic hyperplasia was also detected in transgenic mice expressing Ang1 in the basal epidermal cells. Tie2 was expressed in the lymphatic endothelial cells and Ang1 stimulation of these cells resulted in up-regulation of vascular endothelial growth factor receptor 3 (VEGFR-3). Furthermore, a soluble form of VEGFR-3 inhibited the observed lymphatic sprouting. Our results reinforce the concept that Ang1 therapy may be useful in settings of tissue edema.

Lymphangiogenic growth factors vascular endothelial growth factor (VEGF)-C and VEGF-D have been shown to promote lymphatic metastasis by inducing tumor-associated lymphangiogenesis. In this study, we have investigated how tumor cells gain access into lymphatic vessels and at what stage tumor cells initiate metastasis. We show that VEGF-C produced by tumor cells induced extensive lymphatic sprouting towards the tumor cells as well as dilation of the draining lymphatic vessels, suggesting an active role of lymphatic endothelial cells in lymphatic metastasis. A significant increase in lymphatic vessel growth occurred between 2 and 3 weeks after tumor xenotransplantation, and lymph node metastasis occurred at the same stage. These processes were blocked dose-dependently by inhibition of VEGF receptor 3 (VEGFR-3) signaling by systemic delivery of a soluble VEGFR-3-immunoglobulin (Ig) fusion protein via adenoviral or adeno-associated viral vectors. However, VEGFR-3-Ig did not suppress lymph node metastasis when the treatment was started at a later stage after the tumor cells had already spread out, suggesting that tumor cell entry into lymphatic vessels is a key step during tumor dissemination via the lymphatics. Whereas lymphangiogenesis and lymph node metastasis were significantly inhibited by VEGFR-3-Ig, some tumor cells were still detected in the lymph nodes in some of the treated mice. This indicates that complete blockade of lymphatic metastasis may require the targeting of both tumor lymphangiogenesis and tumor cell invasion.

Platelet-derived growth factor-D (PDGF-D) is a recently characterized member of the PDGF family with unknown in vivo functions. We investigated the effects of PDGF-D in transgenic mice by expressing it in basal epidermal cells and then analyzed skin histology, interstitial fluid pressure, and wound healing. When compared with control mice, PDGF-D transgenic mice displayed increased numbers of macrophages and elevated interstitial fluid pressure in the dermis. Wound healing in the transgenic mice was characterized by increased cell density and enhanced recruitment of macrophages. Macrophage recruitment was also the characteristic response when PDGF-D was expressed in skeletal muscle or ear by an adeno-associated virus vector. Combined expression of PDGF-D with vascular endothelial growth factor-E (VEGF-E) led to increased pericyte/smooth muscle cell coating of the VEGF-E-induced vessels and inhibition of the vascular leakiness that accompanies VEGF-E-induced angiogenesis. These results show that full-length PDGF-D is activated in tissues and is capable of increasing interstitial fluid pressure and macrophage recruitment and the maturation of blood vessels in angiogenic processes.

Background: Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall., Methods: We studied adeno-associated viruses (AAV; 9 x 10(8) to 4 x 10(9) TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 x 10(9) to 1 x 10(10) pfu/ml). 100 microl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments., Results: In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages., Conclusions: Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases., (Copyright 2004 John Wiley & Sons, Ltd.)

Macrophage scavenger receptors (MSR) promote atherosclerotic lesion formation, and modulation of MSR activity has been shown to influence atherosclerosis. Soluble receptors are effective in inhibiting receptor-mediated functions in various diseases. We have generated a secreted macrophage scavenger receptor (sMSR) that consists of the bovine growth hormone signal sequence and the human MSR A I extracellular domains. sMSR reduces degradation of atherogenic modified low-density lipoproteins and monocyte/macrophage adhesion on endothelial cells in vitro. To test long-term effects of sMSR, atherosclerosis-susceptible LDLR knockout mice were transduced via the tail vein with an adeno-associated virus (AAV) expressing sMSR or control enhanced green fluorescent protein (EGFP), and a Western-type diet was started. Gene transfer caused a temporary elevation in alkaline phosphatase and aspartate amino transferase values without a change in C-reactive protein. sMSR protein was detected in the plasma of the transduced mice by a specific ELISA 6 months after the gene transfer. AAV-mediated sMSR gene transfer reduced atherosclerotic lesion area in the aorta by 21% (P < 0.05) compared to EGFP-transduced control mice. Even though eradication of established disease was not possible, atherosclerotic lesion formation could be modified using AAV-mediated gene transfer of the decoy sMSR.

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3-specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF-C/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR-3 gene are associated with hereditary lymphedema. However, VEGF-C can have potent effects on blood vessels because its receptor VEGFR-3 is expressed in certain blood vessels and because the fully processed form of VEGF-C also binds to the VEGFR-2 of blood vessels. To characterize the in vivo effects of VEGF-C on blood and lymphatic vessels, we have overexpressed VEGF-C via adenovirus- and adeno-associated virus-mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose-dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR-2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF-C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF-C or AAV-VEGF-C. These results show that virally produced VEGF-C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.

Primary human lymphedema (Milroy's disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.

Vascular endothelial growth factor receptor 3 (VEGFR-3) is required for cardiovascular development during embryogenesis. In adults, this receptor is expressed in lymphatic endothelial cells, and mutant VEGFR3 alleles have been implicated in human hereditary lymphedema. To better understand the basis of its specific endothelial lineage-restricted expression, we have characterized the VEGFR3 gene and its regulatory 5' flanking region. The human gene contains 31 exons, of which exons 30a and 30b are alternatively spliced. The VEGFR3 proximal promoter is TATA-less and contains stretches of sequences homologous with the mouse Vegfr3 promoter region. In transfection experiments of cultured cells, the Vegfr3 promoter was shown to control endothelial cell-specific transcription of downstream reporter genes. This result was further confirmed in vivo; in a subset of transgenic mouse embryos, a 1.6 kb Vegfr3 promoter fragment directed weak lymphatic endothelial expression of the LacZ marker gene. This suggests that endothelial cell-specific elements occur in the proximal promoter, although further enhancer elements are probably located elsewhere. The sequence, organization, and variation in the VEGFR3 gene and its regulatory region provide important tools for the molecular genetic analysis of the lymphatic system and its disorders.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.

Background: Vascular endothelial growth factor (VEGF) is an important regulator of endothelial cell proliferation, migration, and permeability during embryonic vasculogenesis as well as in physiological and pathological angiogenesis. The recently isolated VEGF-B and VEGF-C cDNAs encode novel growth factor genes of the VEGF family., Methods and Results: Southern blotting and polymerase chain reaction analysis of somatic cell hybrids and fluorescence in situ hybridization (FISH) of metaphase chromosomes were used to assess the chromosomal localization of VEGF-B and VEGF-C genes. The VEGF-B gene was found on chromosome 11q13, proximal to the cyclin D1 gene, which is amplified in a number of human carcinomas. However, VEGF-B was not amplified in several mammary carcinoma cell lines containing amplified cyclin D1. The VEGF-C gene was located on chromosome 4q34, close to the human aspartylglucosaminidase gene previously mapped to 4q34-35., Conclusions: The VEGF-B locus in 11q13 and the VEGF-C locus in 4q34 are candidate targets for mutations that lead to vascular malformations or cardiovascular diseases.

The FLT4, FLT1 and KDR/FLK1 genes encode structurally similar endothelial cell receptor tyrosine kinases. Recently it has been shown that the FLT1 and KDR/FLK-1 proteins function as high-affinity receptors for vascular endothelial growth factor (VEGF). Here we show that FLT4 does not act as a receptor for VEGF, as VEGF did not show specific binding to the FLT4 tyrosine kinase or induce its autophosphorylation. Also, FLT4 did not interact with KDR in response to VEGF. However, when fused with the ligand binding domain of the colony stimulating factor-1 receptor (CSF-1R), the FLT4 tyrosine kinase was specifically activated by CSF-1. The activated FLT4 tyrosine kinase domain was found to interact with the Src homology 2 domains of the SHC and GRB2 adaptor proteins in vitro and with SHC in cells. CSF-1 stimulation of the CSF-1R/FLT4 receptor chimera induced thymidine incorporation in serum-starved NIH3T3 fibroblasts, but not in porcine aortic or murine lung capillary endothelial cells, although tyrosyl phosphorylation of the receptor and SHC occurred in these cells as well. These results suggest that the endothelial cell FLT4 receptor tyrosine kinase transmits signals for an as yet unidentified growth factor.

FLT4 is a recently cloned gene encoding a transmembrane tyrosine kinase related to the FLT1 and KDR/FLK1 vascular endothelial growth factor receptors. We have previously shown that FLT4 is expressed as transcripts of 4.5 and 5.8 kb in several human fetal and adult tissues. Here we show that these transcripts encode two polypeptides, FLT4s (short) and FLT41 (long), which are proteolytically processed in transfected cells and leukemia cells and which have different carboxy terminal tails. The 3' coding region of the 5.8 kb mRNA was found to be 65 codons longer than that of the the 4.5 kb mRNA. Analysis of the genomic structure of the region encoding the two carboxy termini revealed that the two transcripts are generated by alternative polyadenylation and subsequent alternative splicing during RNA processing. Our findings thus show regulation of FLT4 structure in the carboxy terminal tail considered important for receptor function. The significance of the two forms may relate to the role of additional potential autophosphorylation sites in the FLT4 long form.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.

The receptors for at least two hematopoietic growth factors, namely the stem cell factor and colony-stimulating factor 1, belong to class III receptor tyrosine kinases. Here we describe cloning of a partial complementary DNA for FLT4, an additional member of this gene family from human leukemia cells. The FLT4 tyrosine kinase domain is 79% homologous with the previously cloned FLT1 (M. Shibuya et al., Oncogene, 5: 519-524, 1990) tyrosine kinase and maps to the chromosomal region 5q33-qter. We have found FLT4 expression in human placenta, lung, heart, and kidney, whereas the pancreas and brain appeared to contain very little if any FLT4 RNA. The results suggest that FLT4 functions in multiple adult tissues.