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Lymphangiogenesis and Kaposi's Sarcoma VEGF-C


Angiogenesis (blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.

Pat O'Connor

May 23, 2008


Vascular Endothelial Growth Factor-C (VEGF-C) and its Receptors KDR and flt-4 are Expressed in AIDS-Associated Kaposi’s Sarcoma

Journal of Investigative Dermatology 113, 1047-1053 (1999)
© 1999 The Society for Investigative Dermatology

Mihaela Skobe, Lawrence F. Brown*, Kathi Tognazzi*, Ramesh K. Ganju{dagger}, Bruce J. Dezube{dagger}, Kari Alitalo{ddagger} and Michael Detmar

Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, U.S.A.; * Department of Pathology and {dagger} Division of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, U.S.A.; {ddagger} Haartman Institute, University of Helsinki, Finland

Reprint requests to: Dr. Michael Detmar, CBRC/Department of Dermatology, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, U.S.A. Email: michael.detmar@cbrc2.mgh.

Kaposi’s sarcoma is characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis that the lymphangiogenesis factor vascular endothelial growth factor-C and its receptors, KDR and flt-4, are involved in the pathogenesis of Kaposi’s sarcoma, we performed in situ hybridizations and immunofluorescent stainings on human immunodeficiency virus-associated Kaposi’s sarcoma. Spindle-shaped tumor cells strongly expressed KDR and flt-4 mRNA. Immunofluorescent staining confirmed expression of the flt-4 receptor in Kaposi’s sarcoma cells, and double labeling revealed its colocalization with the endothelial cell marker CD31. Vascular endothelial growth factor-C was strongly expressed in blood vessels associated with Kaposi’s sarcoma. In vitro, human dermal microvascular endothelial cells also expressed vascular endothelial growth factor-C mRNA that was further upregulated by vascular permeability factor/vascular endothelial growth factor. Vascular endothelial growth factor-C potently stimulated the proliferation of Kaposi’s sarcoma tumor cells in vitro. These results demonstrate important paracrine functions of vascular endothelial growth factor-C, produced by blood vessels, in the pathogenesis of cutaneous Kaposi’s sarcoma, and suggest a lymphatic origin and/or differentiation of Kaposi’s sarcoma tumor cells.

Key Words: angiogenesis • HIV • lymphatics • VEGF.

Abbreviations: HDMEC, human dermal microvascular endothelial cells ISH, in situ hybridization KS, Kaposi’s sarcoma VEGF-C, vascular endothelial growth factor-C


Kaposi’s sarcoma (KS) is the most common tumor associated with AIDS, frequently arising in the skin and mucosal surfaces (Enzinger & Weiss 1995; Regezi et al. 1993). Histologically, KS lesions are composed of clusters of spindle-shaped tumor cells, blood vessels, fibroblasts, dendrocytes, and inflammatory cells (Enzinger & Weiss 1995; Naidu et al. 1994). The principal features of AIDS-KS include hyperproliferation of spindle cells, prominent angiogenesis, increased vascular permeability, edema, and extravasation of erythrocytes (Roth et al. 1992; Sturzl et al. 1992).

The etiology of KS is still a subject of dispute. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. Already in 1902 it was proposed that the vascular slits comprising the lesions are morphologically consistent with lymphatics, based on the spindle shape of their lining cells, their tenuous basement membrane, and the scarcity of red blood cells in their vascular lumen (Philippson 1902; Pelagatti 1905). Subsequent ultrastructural and immunohistochemical evidence largely supported the view that the KS tumor cells more closely resemble lymphatic than blood vascular endothelium (Beckstead et al. 1985; Dorfman 1962; Dictor 1986; Witte et al. 1990). Further patterns suggestive of a lymphatic derivation of KS are the unique distribution of cutaneous lesions along the lines of lymphatics, their arrangement around blood vessels, and the absence of lesions in organs that are devoid of lymphatics (Dorfman 1988).

It has been recently proposed that angiogenesis and vascular permeability play a central part in the development of KS, strongly suggesting a role for vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in its pathogenesis. VPF/VEGF stimulates endothelial cell growth in vitro (Detmar et al. 1995) and induces angiogenesis in vivo (Oh et al. 1997), acting through two tyrosine kinase receptors that are predominantly found on vascular endothelial cells: flt-1 (VEGFR-1) and KDR (VEGFR-2) (Neufeld et al. 1999). Previously, we reported strong expression of KDR mRNA in KS tumor cells as well as in endothelial cells of small vessels within and surrounding the tumor; however, we did not detect significant expression of the VEGF receptor flt-1 in spindle-shaped tumor cells (Brown et al. 1996). These findings have been confirmed by others, suggesting an important function for KDR in the development of KS (Cornali et al. 1996; Masood et al. 1997). It has remained controversial, however, whether VPF/VEGF itself is expressed in KS tumor cells. Expression of VPF/VEGF has been reported in KS cell lines in vitro and in vivo (Weindel et al. 1992; Cornali et al. 1996; Masood et al. 1997; Nakamura et al. 1997). Anti-sense oligonucleotides to VPF/VEGF inhibited the growth of KS cells in vitro; however, addition of VPF/VEGF did not result in increased proliferation of KS cells (Cornali et al. 1996; Masood et al. 1997). Moreover, we previously found VPF/VEGF predominantly expressed by epidermal keratinocytes overlying KS tumors and, at lower levels, by infiltrating inflammatory cells, but only weakly by KS cells (Brown et al. 1996). The strong expression of KDR, but only weak expression of VPF/VEGF suggested that some other ligand(s) of KDR might be involved in the pathogenesis of KS.

Recently, VEGF-C has been identified as a new member of the VEGF family of growth factors (Joukov et al. 1996; Lee et al. 1996). Like VPF/VEGF, VEGF-C binds to KDR; however, it does not bind to the VPF/VEGF receptor flt-1 (Joukov et al. 1996). VEGF-C also activates the receptor tyrosine kinase flt-4 (VEGFR-3), that has been regarded as a specific marker for lymphatic endothelium (Kaipainen et al. 1995; Joukov et al. 1996). Expression of flt-4 was very recently reported in KS cells (Liu et al. 1997; Jussila et al. 1998). VEGF-C elicits a lymphangiogenic response in the chicken embryo chorioallantoic membrane (CAM), and transgenic mice overexpressing VEGF-C in the skin are characterized by specific hyperplasia of the lymphatic network, revealing VEGF-C as the first known growth factor for lymphatic endothelium (Jeltsch et al. 1997; Oh et al. 1997).

In this study we examined the expression of VEGF-C, VPF/VEGF and their receptors in AIDS-associated KS, and characterized the effects of VEGF-C on KS tumor cells, in order to test the hypothesis that the lymphangiogenesis factor VEGF-C and its receptors are involved in the pathogenesis of KS.


In situ hybridization (ISH)
A VEGF-C cDNA fragment comprising nt 827–1634 of the full length human VEGF-C cDNA (Joukov et al. 1996; Lee et al. 1996) (GenBank accession no. X94216) was subcloned into the pGEM-3Z vector (Promega, Madison, WI). Following restriction digestion with AccI and ClaI, a 808 bp fragment was gel purified and ligated into the AccI site of the pGEM-3Z vector. The sequence was verified by restriction mapping and by direct sequencing using the Sanger dideoxy method. For generation of anti-sense and sense probes, the construct was linearized with BamHI or HindIII and transcribed from the SP6or T7promoter, respectively. The human flt-4 cDNA used for generation of ISH probes comprised nt 55–207 of the full length flt-4 cDNA, and has been published previously (Aprelikova et al. 1992; Pajusola et al. 1992). 35S-labeled single-stranded anti-sense and sense RNA probes for the VPF/VEGF receptors KDR and flt-1 have been described previously (Detmar et al. 1994). The probe for human VPF/VEGF hybridizes with a region of VPF/VEGF mRNA common to all known VPF/VEGF splice variants (Brown et al. 1992).

ISH was performed on 4 µm-thick sections of paraffin-embedded tissue as described in detail previously (Brown et al. 1996). Six cases of AIDS-associated cutaneous KS were studied. Four millimeter punch biopsies of cutaneous KS lesions were obtained with informed consent from HIV-positive KS patients following human experimental guidelines of the US Department of Health and Human Services and the Beth Israel Deaconess Medical Center. For autoradiography, slides were coated with NTB2 film emulsion and exposed for 2 wk. The slides were developed and counterstained with hematoxylin. Specimens were viewed using a Nikon E-600 microscope (Nikon, Melville, NY).

Indirect immunofluorescence
Human tissues obtained after surgical removal were immediately frozen in liquid nitrogen, sectioned (6 µm), stored at - 70°C, and used for immunofluorescence. Cryosections were fixed and stained as previously described (Skobe et al. 1997), using antibodies against human CD31 (dilution 1/30; Pharmingen, San Diego, CA), human flt-4 (1.1 µg per ml) (Jussila et al. 1998) or anti-serum against human VEGF-C (cl.882/5; dilution 1/100) (Joukov et al. 1997). The secondary antibodies, labeled with either Texas Red or fluorescein isothiocyanate (Jackson ImmunoResearch Lab., West Grove, PA) were used at 30 µg per ml. Specimens were mounted in Mowiol (Calbiochem, La Jolla, CA) and examined by confocal imaging using a Leica DM IRBE microscope and a Leica TCS 4D confocal system.

Kaposi’s sarcoma cell growth assays
The human KS cell line KS59, derived from a cutaneous biopsy of an AIDS patient, has been previously shown to express the flt-4 receptor that was phosphorylated after treatment with VEGF-C (Liu et al. 1997). Cells were plated in triplicate in collagen-coated 24 well plates at a density of 2 x 103 cells per well in complete medium containing 15% fetal bovine serum (FBS) (Liu et al. 1997). Cells were allowed to attach overnight, washed 3 x with serum-free medium, and were cultured in medium containing 0.5% FBS and 1–100 ng per ml recombinant human VEGF-C (Joukov et al. 1997). Medium and VEGF-C were replaced after 2 d. After 4 d of treatment, cells were trypsinized and counted using a Coulter Counter. The experiment was repeated twice with comparable results. The unpaired t test was used for statistical analysis of the results.

Growth factor stimulation of endothelial cells and northern blot analysisHuman dermal microvascular endothelial cells (HDMEC) were isolated from neonatal foreskins as described (Richard et al. 1998) and were used between passage 4 and 6. HDMEC were cultured in endothelial cell basal medium (Clonetics, San Diego, CA), supplemented with 1 µg per ml hydrocortisone acetate, 5 x 10- 5 M N-6,2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (Sigma, St. Louis, MO), 20% heat-inactivated FBS, 100 U per ml penicillin, and 100 µg per ml streptomycin, on collagen-coated tissue culture dishes (Richard et al. 1998) until the cells reached 80% confluency. Cultures were then switched to 2% FBS for 2 h and were treated with growth factors (0.1–100 ng per ml) for 48 h. Recombinant human VPF/VEGF165, platelet-derived growth factor (PDGF)-AA, PDGF-BB, basic fibroblast growth factor, placenta growth factor (PCGF) and tumor necrosis factor (TNF)- were all purchased from R&D (Minneapolis, MN). Total cellular RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The isolated RNA was subjected to electrophoresis and transferred to Hybond-N + nylon supported membranes (Amersham, Arlington Heights, IL). 32P-radiolabeled DNA probes were labeled by the random priming method (Multiprime Labeling Kit, Amersham). We used a fragment containing nt 581–1634 of human VEGF-C cDNA (Joukov et al. 1996; Lee et al. 1996) (GenBank accession no. X94216). A human ß-actin cDNA probe (Clontech, Palo Alto, CA) was used as the control for equal RNA loading. Blots were hybridized at 65°C for 24 h, washed at high stringency and exposed to X-OMAT MR film (Kodak, Rochester, NY). All experiments were repeated twice.

Endothelial cell proliferation assays
Proliferation assays were performed as described (Detmar et al. 1995). Briefly, HDMEC were seeded at 1 x 104 cells per well into collagen-coated 24-well tissue culture clusters in complete endothelial cell basal medium supplemented with 20% FBS. The following day, at approximately 30–40% confluency, cultures were switched to 2% FBS for 24 h. HDMEC were then incubated with various concentrations of VEGF-C (0.3–30 ng per ml) and/or VPF/VEGF (0.4 ng per ml) for 96 h. Medium and growth factors were replaced after 48 h. [3H]thymidine was added to the cultures 6 h before harvesting (1 µCi per ml) (DuPont NEN, Boston, MA) and incorporated radioactivity was determined as described (Detmar et al. 1989). All experiments were performed in triplicate, and were repeated twice. The unpaired t test was used for statistical analysis of the results.


Expression of VEGF-C, VPF/VEGF, and their receptors in AIDS-associated KS
To investigate whether VEGF-C and its receptors might be involved in the development of AIDS-associated KS, we performed ISH on six cases of cutaneous KS. The specificity of all ISH reactions was demonstrated by hybridization with the radio-labeled sense RNA probes, which did not reveal any signal. High amounts of KDR mRNA (receptor for both, VEGF-C and VPF/VEGF) were detected in numerous spindle-shaped cells in all AIDS-KS lesions examined (Fig. 1A,B). Endothelial cells of small blood vessels within and immediately adjacent to the lesions labeled strongly for both, KDR and flt-1 mRNA (Fig. 1A-D, respectively), as previously reported (Brown et al. 1996). Tumor cells, however, expressed little or no flt-1 mRNA (Fig. 1C,D). Importantly, hybridization analysis showed that flt-4 mRNA was abundantly expressed in spindle cells of AIDS-KS (Fig. 1E,F). In contrast, endothelial cells lining small blood vessels expressing KDR and flt-1 mRNA did not express any flt-4 mRNA (Fig. 1E,F). Immunofluorescent staining confirmed expression of flt-4 protein in spindle-shaped KS cells, and double immuno- fluorescence labeling revealed its colocalization with the endothelial cell marker CD31 (Fig. 2). Whereas KS tumor cells did not express any significant amounts of VPF/VEGF (Fig. 1I, J) or VEGF-C mRNA, VEGF-C mRNA was expressed at high levels in endothelial cells of arteries as well as of small blood vessels associated with KS lesions (Fig. 1G,H). The expression of VEGF-C protein in blood vessels associated with KS lesions was confirmed by immunofluorescent stainings (Fig. 3). Occasionally, cells closely apposed to endothelial cells, most likely smooth muscle cells, also labeled strongly for VEGF-C protein (Fig. 3).

In summary, whereas endothelial cells of small blood vessels labeled strongly for KDR and flt-1, but not for flt-4 mRNA, the KS tumor cells expressed KDR and flt-4, but not flt-1 mRNA. Furthermore, VEGF-C, the ligand for KDR and flt-4, was found to be expressed in KS lesions, suggesting a role of VEGF-C and its receptors in the pathogenesis of AIDS-associated KS.

VEGF-C stimulates proliferation of KS cells in vitro
The expression of the flt-4 receptor in KS tumor cells and of its ligand VEGF-C in endothelial cells of KS lesions suggested a paracrine role of VEGF-C in KS tumor growth. Therefore, we tested the capability of VEGF-C to promote the proliferation of cultured KS cells which are known to express the flt-4 receptor (Liu et al. 1997). VEGF-C induced a potent, concentration-dependent increase in cell numbers after 4 d of treatment with 1–100 ng of human recombinant VEGF-C (Fig. 4), revealing VEGF-C as a growth factor for KS tumor cells.

VPF/VEGF upregulates expression of VEGF-C in vascular endothelial cells
As we observed increased levels of VEGF-C mRNA in endothelial cells of blood vessels surrounding cutaneous KS lesions, we next studied the regulation of VEGF-C gene expression in HDMEC (Richard et al. 1998) by angiogenic growth factors. HDMEC at 80% confluency were treated with endothelial growth factors for 4–24 h, after which total RNA was isolated, electrophoresed, and subjected to northern blot hybridization using a human VEGF-C probe. Moderate amounts of steady-state VEGF-C mRNA were detectable in HDMEC under basal conditions (Fig. 5A). Northern hybridization revealed a single VEGF-C band in HDMEC; however, a faint, slower migrating second band was observed after longer exposure times. Following treatment with VPF/VEGF, a concentration-dependent increase in VEGF-C mRNA expression was seen after 4 h, continuing throughout the experiment (24 h). Maximal stimulation was obtained with 20 ng per ml VPF/VEGF (Fig. 5A). In contrast, induction of VEGF-C expression in HDMEC was not observed following treatment with basic fibroblast growth factor, PDGF-AA, PDGF-BB, PlGF, or TNF- (data not shown).

VEGF-C stimulates proliferation of HDMEC
To test the hypothesis that VEGF-C could be an autocrine growth factor for vascular endothelial cells, we next examined the ability of VEGF-C to stimulate the proliferation of microvascular endothelial cells derived from the skin. HDMEC were treated with VEGF-C (0.3–30 ng per ml) alone or in combination with VPF/VEGF (0.4 ng per ml). Stimulation of [3H]thymidine incorporation was measured after 48 h of treatment. VEGF-C induced a concentration-dependent increase in [3H]thymidine incorporation by HDMEC (Fig. 5B), with a minimal effective concentration of 0.3 ng per ml. The extent of stimulation by VEGF-C was comparable with that of VPF/VEGF in all concentrations tested (data not shown). When administered together with VPF/VEGF, the effect of VEGF-C was markedly increased as compared with the effect observed with each factor alone. In fact, in a concentration range where each of the factors had little effect (0.3 ng per ml), co-addition of both factors induced synergistic effects. With a moderate increase in VEGF-C concentration (3 ng per ml), the response was greater than additive. These results identify VEGF-C as a potent mitogenic factor for skin microvascular endothelial cells.


VEGF-C is a novel member of the VEGF family of angiogenic growth factors, distinguished by its capacity to stimulate growth of lymphatic vascular endothelium in vivo (Joukov et al. 1996; Lee et al. 1996; Jeltsch et al. 1997; Oh et al. 1997). In adult human tissues, the VEGF-C receptor flt-4 has been found exclusively expressed by lymphatic endothelium, and thus has been considered a marker of lymphatic vessels (Jussila et al. 1998; Kaipainen et al. 1995). The second VEGF-C receptor, KDR, is prevalently expressed by activated endothelium of blood vessels, and is also utilized by VPF/VEGF (Joukov et al. 1996; Neufeld et al. 1999). Previously, we found strong expression of KDR in KS tumor cells (Brown et al. 1996). Surprisingly however, we were unable to detect high levels of its ligand VPF/VEGF in most cases (Brown et al. 1996), prompting us to examine the hypothesis that a different KDR ligand, VEGF-C, may partake in the development of KS.

In this study we show that VEGF-C mRNA and protein were strongly expressed by endothelial cells of large blood vessels surrounding the KS lesions and that high levels of flt-4 receptor mRNA and protein were expressed in spindle-shaped KS tumor cells. In agreement with our findings, the expression of flt-4 protein has very recently been reported in KS spindle cells in vivo (Jussila et al. 1998; Weninger et al. 1999). VEGF-C has also been shown to activate the flt-4 receptor present on cultured KS cells (Liu et al. 1997). We demonstrate here that VEGF-C is a potent mitogen for KS tumor cells in vitro. Taken together, our results strongly suggest that VEGF-C expressed by endothelial cells stimulates proliferation of KS tumor cells in vivo. It remains to be established whether another flt-4 ligand, VEGF-D (Achen et al. 1998) or some yet unidentified ligands may also be implicated.

VEGF-C may further play a part in KS through interaction with the KDR receptor, which was strongly expressed by KS tumor cells, in accordance with previous studies (Brown et al. 1996; Masood et al. 1997). Based on the expression of VPF/VEGF by cultured KS cells (Weindel et al. 1992; Cornali et al. 1996; Masood et al. 1997; Nakamura et al. 1997), VPF/VEGF has been suggested as a factor of paramount importance in KS. The data regarding the expression of VPF/VEGF in KS cells in vivo, however, remained inconclusive. Here we confirm our earlier findings that VPF/VEGF was not consistently expressed by KS tumor cells. Whereas we identified inflammatory cells infiltrating KS lesions and hyperplastic keratinocytes as alternate sources of VPF/VEGF (Brown et al. 1996), the question remains whether the levels produced by these cells are sufficient to bring about effects such as proliferation of KS tumor cells, prominent angiogenesis and vascular permeability. It appears that VEGF-C present in KS may function either in cooperation or in competition with VPF/VEGF for the KDR receptor. To test these possibilities, we examined the effects of VEGF-C alone or in combination with VPF/VEGF on human skin microvascular endothelial cells in culture. VEGF-C promoted proliferation of HDMEC with a similar potency as VPF/VEGF. In contrast, it has been reported that VEGF-C was 50–100-fold less potent than VPF/VEGF in inducing the proliferation of bovine capillary endothelial cells (Joukov et al. 1996; Joukov et al. 1997), human umbilical vein endothelial cells (HUVEC), or lung microvascular endothelial cells (Lee et al. 1996; Witzenbichler et al. 1998). When injected s.c. into the skin however, VEGF-C was only 4–5-fold less potent in inducing microvascular permeability (Joukov et al. 1997), suggesting that endothelial cells derived from the skin may be more responsive to VEGF-C than endothelial cells derived from other body sites. When administered together with VPF/VEGF, the effect of VEGF-C on endothelial cell proliferation was clearly enhanced. In a concentration range where each of the factors had very little effect, the co-addition induced synergistic effects. With a moderate increase in VEGF-C concentration, the response became additive. These data indicate that, if the same synergy exists in vivo, even very low concentrations of both growth factors might have pronounced effects, possibly sufficient to induce neovascularization and vascular permeability. Thus, VEGF-C, in cooperation with VPF/VEGF, may play a part in KS tumor cell proliferation, angiogenesis, and edema typically seen in these tumors. In accordance with our findings, a recent report demonstrated synergism between VEGF-C and basic fibroblast growth factor or VPF/VEGF on in vitro angiogenesis (Pepper et al. 1998). The mitogenic effect of VEGF-C on cultured endothelial cells is probably mediated through KDR, as HDMEC did not express significant amounts of the flt-4 receptor. Indeed, a recombinant point mutant of VEGF-C that binds and activates selectively flt-4 did not induce endothelial cell migration and vascular permeability (Joukov et al. 1998). Expression of flt-4 mRNA in cultured human endothelial cells has been observed by others (Hewett & Murray 1996), possibly reflecting a small number of lymphatic endothelial cells contained within cultured blood vascular endothelial cells. Alternatively, the flt-4 expression pattern may be modulated in culture and may not reflect the expression pattern observed in vivo.

Thus far, expression of VEGF-C by endothelial cells in vivo had not been reported. We observed significant amounts of VEGF-C mRNA and protein expressed by blood vascular endothelium adjacent to KS lesions in vivo and have verified this finding using HDMEC in vitro. Cultured HUVEC were recently found to express VEGF-C mRNA, and its levels were increased by treatment with interleukin-1ß and TNF- (Ristimaki et al. 1998). As outlined above, VEGF-C greatly stimulated proliferation of HDMEC in vitro, raising the intriguing possibility that VEGF-C, upregulated in vascular endothelium under certain conditions, may regulate endothelial cell responses in an autocrine manner.

There are significant differences in the regulation of VPF/VEGF and VEGF-C gene transcription. Cytokines and growth factors induce VEGF-C mRNA expression in cultured cells yet hypoxia and oncogenes, important regulators of VPF/VEGF expression, have no effect (Enholm et al. 1997). In human fibroblasts, VEGF-C transcription is stimulated by serum and its components, and by the inflammatory cytokines interleukin-1, interleukin-1ß, and TNF- (Enholm et al. 1997; Ristimaki et al. 1998). We studied the regulation of the VEGF-C gene expression by growth factors in endothelial cells. Importantly, VPF/VEGF induced strong, concentration-dependent stimulation of VEGF-C transcription, whereas no effects were observed upon stimulation with the other growth factors tested, i.e., PDGF, basic fibroblast growth factor, PlGF, and TNF-. Thus, VPF/VEGF may induce endothelial cell responses in part by inducing a VEGF-C autocrine loop. Furthermore, the regulation of the expression of the lymphangiogenic factor VEGF-C by the major angiogenic factor VPF/VEGF is an interesting concept, as it is conceivable that tissues with increased demands for angiogenesis would also increase their demands for lymphangiogenesis.

In conclusion, our results suggest that VEGF-C and its receptors flt-4 and KDR play a major part in the pathogenesis of AIDS-associated KS. In cooperation, VEGF-C and VPF/VEGF are likely to be involved in KS tumor formation by stimulating tumor cell proliferation, angiogenesis, and microvascular permeability. The strong expression of the flt-4 receptor in KS tumor cells, its coexpression with the endothelial cell marker CD31, and the presence of the lymphangiogenic factor VEGF-C within the lesions are in support of a lymphatic origin and/or differentiation of KS tumor cells.


This work was supported by the Human Frontier Science Program (MS), by NIH/NCI grant CA69184 (MD), by American Cancer Society Research Project grant 99-23901 (MD), and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (MD).


Achen, MG, Jeltsch, M, Kukk, E et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci USA, 95, 548–553, 1998.[Abstract/Free Full Text]

Aprelikova, O, Pajusola, K, Partanen, J et al. FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res, 52, 746–748, 1992.[Abstract]

Beckstead, JH, Wood, GS & Fletcher, V. Evidence for the origin of Kaposi’s sarcoma from lymphatic endothelium. Am J Pathol, 119, 294–300, 1985.[Abstract]

Brown, LF, Berse, B, Tognazzi, K et al. Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int, 42, 1457–1461, 1992.[Medline]

Brown, LF, Tognazzi, K, Dvorak, HF & Harrist, TJ. Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi’s sarcoma and cutaneous angiosarcoma. Am J Pathol, 148, 1065–1074, 1996.[Abstract]

Cornali, E, Zietz, C, Benelli, R et al. Vascular endothelial growth factor regulates angiogenesis and vascular permeability in Kaposi’s sarcoma. Am J Pathol, 149, 1851–1869, 1996.[Abstract]

Detmar, M, Mayer -d, a-Silva, A, Stadler, R & Orfanos, CE. Effects of azelaic acid on proliferation and ultrastructure of mouse keratinocytes in vitro. J Invest Dermatol, 93, 70–74, 1989.[Abstract]

Detmar, M, Brown, LF, Claffey, KP et al. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med, 180, 1141–1146, 1994.[Abstract]

Detmar, M, Yeo, K-T, Nagy, JA et al. Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells. J Invest Dermatol, 105, 44–50, 1995.[Abstract]

Dictor, M. Kaposi’s sarcoma. Origin and significance of lymphaticovenous connections. Virchows Arch a Pathol Anat Histopathol, 409, 23–35, 1986.[Medline]

Dorfman, RF. Kaposi’s sarcoma: The contribution of enzyme histochemistry to the identification of cell types. Acta Int Union Cancer, 18, 464–476, 1962.

Dorfman, RF. Kaposi’s sarcoma: Evidence supporting its origin from the lymphatic system. Lymphology, 21, 45–52, 1988.[Medline]

Enholm, B, Paavonen, K, Ristimaki, A et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia. Oncogene, 14, 2475–2483, 1997.[Medline]

Enzinger, FM & Weiss, SW. Soft Tissue Tumors. St Louis, MO. Mosby, 1995.

Hewett, PW & Murray, JC. Coexpression of flt-1, flt-4 and KDR in freshly isolated and cultured human endothelial cells. Biochem Biophys Res Commun, 221, 697–702, 1996.[Medline]

Jeltsch, M, Kaipainen, A, Joukov, V et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science, 276, 1423–1425, 1997.[Abstract/Free Full Text]

Joukov, V, Pajusola, K, Kaipainen, A et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J, 15, 1751, 1996.[Medline]

Joukov, V, Sorsa, T, Kumar, V et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J, 16, 3898–3911, 1997.[Abstract/Free Full Text]

Joukov, V, Kumar, V, Sorsa, T, Arighi, E, Weich, H, Saksela, O & Alitalo, K. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem, 273, 6599–6602, 1998.[Abstract/Free Full Text]

Jussila, L, Valtola, R, Partanen, TA et al. Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res, 58, 1599–1604, 1998.[Abstract]

Kaipainen, A, Korhonen, J, van Mustonen, THV, Fang, GH, Dumont, D, Breitman, M & Alitalo, K. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA, 92, 3566–3570, 1995.[Abstract]

Lee, J, Gray, A, Yuan, J, Luoh, SM, Avraham, H & Wood, WI. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc Natl Acad Sci USA, 93, 1988–92, 1996.[Abstract/Free Full Text]

Liu, ZY, Ganju, RK, Wang, JF et al. Cytokine signaling through the novel tyrosine kinase RAFTK in Kaposi’s sarcoma cells. J Clin Invest, 99, 1798–1804, 1997.[Abstract/Free Full Text]

Masood, R, Cai, J, Zheng, T, Smith, DL, Naidu, Y & Gill, PS. Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci USA, 94, 979–984, 1997.[Abstract/Free Full Text]

Naidu, YM, Rosen, EM, Zitnick, R et al. Role of scatter factor in the pathogenesis of AIDS-related Kaposi sarcoma. Proc Natl Acad Sci USA, 91, 5281–5285, 1994.[Abstract]

Nakamura, S, Murakami, MK, Rao, N, Weich, HA & Rajeev, B. Vascular endothelial growth factor is a potent angiogenic factor in AIDS-associated Kaposi’s sarcoma-derived spindle cells. J Immunol, 158, 4992–5001, 1997.[Abstract]

Neufeld, G, Cohen, T, Gengrinovitch, S & Poltorak, Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J, 13, 9–22, 1999.[Abstract/Free Full Text]

Oh, SJ, Jeltsch, MM, Birkenhaeger, R et al. VEGF and VEGF-C: Specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev Biol, 188, 96–109, 1997.[Medline]

Pajusola, K, Aprelikova, O, Korhonen, J, Kaipainen, A, Pertovaara, L, Alitalo, R & Alitalo, K. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res, 52, 5738–5743, 1992.[Abstract]

Pelagatti, M. Sul sarcoma molteplice primitivo del Kaposi. Gior Ital Mal Ven, 40, 569, 1905.

Pepper, MS, Mandriota, SJ, Jeltsch, M, Kumar, V & Alitalo, K. Vascular endothelial growth factor (VEGF) -C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity. J Cell Physiol, 177, 439–452, 1998.[Medline]

Philippson, L. Uber das sarcoma idiopathicum cutis Kaposi. Ein Beitrag zur Sarkomlehre. Virchows Arch Pathol Anat, 167, 58, 1902.

Regezi, JA, MacPhail, LA, Daniels, TE, DeSouza, YG, Greenspan, JS & Greenspan, D. Human immunodeficiency virus-associated oral Kaposi’s sarcoma. A heterogeneous cell population dominated by spindle-shaped endothelial cells. Am J Pathol, 143, 240–249, 1993.[Abstract]

Richard, L, Velasco, P & Detmar, M. A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells. Exp Cell Res, 240, 1–6, 1998.[Medline]

Ristimaki, A, Narko, K, Enholm, B, Joukov, V & Alitalo, K. Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C. J Biol Chem, 273, 8413–8418, 1998.[Abstract/Free Full Text]

Roth, WK, Brandstetter, H & Sturzl, M. Cellular and molecular features of HIV-associated Kaposi’s sarcoma. AIDS, 6, 895–913, 1992.[Medline]

Skobe, M, Rockwell, P, Goldstein, N, Vosseler, S & Fusenig, NE. Halting angiogenesis suppresses carcinoma cell invasion. Nat Med, 3, 1222–1227, 1997.[Medline]

Sturzl, M, Brandstetter, H & Roth, WK. Kaposi’s sarcoma: a review of gene expression and ultrastructure of KS spindle cells in vivo. AIDS Res Hum Retroviruses, 8, 1753–1763, 1992.[Medline]

Weindel, K, Marme, D & Weich, HA. AIDS-associated Kaposi’s sarcoma cells in culture express vascular endothelial growth factor. Biochem Biophys Res Commun, 183, 1167–1174, 1992.[Medline]

Weninger, W, Partanen, TA, Breiteneder-Geleff, S et al. Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi’s sarcoma tumor cells. Lab Invest, 79, 243–251, 1999.[Abstract]

Witte, MH, Witte, CL, Way, DL & Fiala, M. AIDS, Kaposi sarcoma, and the lymphatic system: update and reflections. Lymphology, 23 (2), 73–80, 1990.[Medline]

Witzenbichler, B, Asahara, T, Murohara, T et al. Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol, 153, 381–394, 1998.[Abstract/Free Full Text]

Manuscript received 24 March 1999; revised 26 August 1999; accepted for publication 6 September 1999



Kaposi Sarcoma, KSHV and lymphangiogensis


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The time has come for families, parents, caregivers to have a support group of their own. Support group for parents, families and caregivers of chilren with lymphedema. Sharing information on coping, diagnosis, treatment and prognosis. Sponsored by Lymphedema People.



Lipedema Lipodema Lipoedema

No matter how you spell it, this is another very little understood and totally frustrating conditions out there. This will be a support group for those suffering with lipedema/lipodema. A place for information, sharing experiences, exploring treatment options and coping.

Come join, be a part of the family!




If you are a man with lymphedema; a man with a loved one with lymphedema who you are trying to help and understand come join us and discover what it is to be the master instead of the sufferer of lymphedema.



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Support group for parents, patients, children who suffer from all forms of lymphangiectasia. This condition is caused by dilation of the lymphatics. It can affect the intestinal tract, lungs and other critical body areas.



Lymphatic Disorders Support Group @ Yahoo Groups

While we have a number of support groups for lymphedema... there is nothing out there for other lymphatic disorders. Because we have one of the most comprehensive information sites on all lymphatic disorders, I thought perhaps, it is time that one be offered.


Information and support for rare and unusual disorders affecting the lymph system. Includes lymphangiomas, lymphatic malformations, telangiectasia, hennekam's syndrome, distichiasis, Figueroa
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Lymphedema People New Wiki Pages

Have you seen our new “Wiki” pages yet?  Listed below are just a sample of the more than 140 pages now listed in our Wiki section. We are also working on hundred more.  Come and take a stroll! 

Lymphedema Glossary 


Arm Lymphedema 

Leg Lymphedema 

Acute Lymphedema 

The Lymphedema Diet 

Exercises for Lymphedema 

Diuretics are not for Lymphedema 

Lymphedema People Online Support Groups 



Lymphedema and Pain Management 

Manual Lymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT) 

Infections Associated with Lymphedema 

How to Treat a Lymphedema Wound 

Fungal Infections Associated with Lymphedema 

Lymphedema in Children 


Magnetic Resonance Imaging 

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Axillary node biopsy

Sentinel Node Biopsy

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Magnetic Resonance Imaging 

Lymphedema Gene FOXC2

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