Immunomodulatory effect of vitamin D and its potential role in the prevention and treatment of thyroid autoimmunity: a narrative review.

The main role of vitamin D is to control mineral homeostasis. However, recent studies suggested the existence of a number of extraskeletal effects. Among the latter, preclinical studies provided consistent data on the involvement of vitamin D in innate and adaptive immunity and autoimmunity. Molecular biology studies showed that both vitamin D receptor and vitamin D enzymatic complexes are expressed in a large number of cells and tissues unrelated to mineral homeostasis. In contrast, only a few randomized clinical trials in humans investigated the possible role of vitamin D in the prevention or treatment of immunological disorders. In this regard, low serum vitamin D levels have been reported in observational trials in human autoimmune disorders. The aim of the present paper was to review the potential implications of vitamin D in immune modulation, with special focus on thyroid autoimmune disorders.

NET amyloidogenic backbone in human activated neutrophils.

Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

Growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates.

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Cytokines and chemokines as regulators of angiogenesis in health and disease.

The intricate interplay between the endothelium and immune cells has been well recognized in the context of immune responses. However, the fact that this inter-relation extends well beyond immune regulation is becoming increasingly recognized, with particular regards to the influence of the immune system on the essential endothelial process of angiogenesis, where the contribution of cytokines drives the angiogenic process. As angiogenesis is an important component of numerous pathological states, among these chronic inflammatory conditions and cancer, understanding the role of cytokines and chemokines in guiding new vessel formation provides key insight into novel therapeutic modalities. Here we review the actions of principal cytokines and chemokines on the angiogenic process and discuss how both can be considered potential pharmaceutical targets or pharmaceuticals themselves for modulation of angiogenesis in chronic inflammation associated with cancer, rheumatoid arthritis and other inflammatory diseases.

Inhibition of angiogenesis-driven Kaposi's sarcoma tumor growth in nude mice by oral N-acetylcysteine.

The thiol N-acetyl-L-cysteine (NAC), an analogue and precursor of reduced glutathione, has cancer chemopreventive properties attributable to its nucleophilicity, antioxidant activity, and a variety of other mechanisms. We demonstrated recently that NAC has anti-invasive, antimetastatic, and antiangiogenic effects in in vitro and in vivo test systems. In the present study, s.c. transplantation of KS-Imm cells in (CD-1)BR nude mice resulted in the local growth of Kaposi's sarcoma, a highly vascularized human tumor. The daily administration of NAC with drinking water, initiated after the tumor mass had become established and detectable, produced a sharp inhibition of tumor growth, with regression of tumors in half of the treated mice along with a markedly prolonged median survival time. The production of vascular endothelial growth factor (VEGF) and certain proliferation markers (proliferating cell nuclear antigen and Ki-67) were significantly lower in Kaposi's sarcomas from NAC-treated mice than from control mice. Treatment of KS-Imm cells with NAC in vitro resulted in a dose-dependent inhibition of chemotaxis and invasion through inhibition of gelatinase-A (matrix metalloproteinase-2, MMP-2) activity without altering MMP-2 or MMP-9 mRNA levels. NAC also significantly inhibited VEGF production but did not affect proliferation markers in vitro. Reverse transcription-PCR analysis indicated that total VEGF mRNAs were reduced by 10 mM NAC. Taken together, these findings provide evidence that NAC, the safety of which even at high doses has been established in almost 40 years of clinical use, in addition to its chemopreventive action, has a strong antiangiogenic potential that could be exploited for preventing cancer progression as well as used in cancer adjuvant therapy.

Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi's sarcoma cells.

Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi's sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

RB1 oncosuppressor gene over-expression inhibits tumor progression and induces melanogenesis in metastatic melanoma cells.

The retinoblastoma gene (RB1) is frequently deleted or mutated in many tumor types and in all cases of retinoblastoma. Apart from its role in regulation of the cell cycle, the RB1 gene product (p110RB1) appears to be involved in control of differentiation. Malignant metastatic cells show many properties of poorly differentiated cells, and are highly invasive in vitro and in vivo. We have transfected the human RB1 cDNA in an expression vector under the control of the beta-actin promoter into B16F10 murine melanoma cells. These cells highly overexpress RB1 mRNA and the p110RB1 product, show reduced growth rate and increased melanogenesis in vitro. Vector control transfectants showed no alteration of invasiveness. The p110RB1 over-expressing cells also had a reduced capacity to migrate and invade through an artificial basement membrane, key characteristics of metastatic cells. When injected into nude mice, the p110RB1 over-expressing cells showed reduced tumor growth and reduced metastatic potential. The few metastasis observed were predominantly melanotic. These data indicate that RB1 gene expression is involved in melanoma cell differentiation and plays a role in downregulation of migration, invasion and metastatic potential of these cells.

Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2.

OBJECTIVE: To determine the neoplastic nature of Kaposi's sarcoma (KS). A highly vascularized lesion, KS is frequently associated with AIDS, indicating HIV products may be involved. DESIGN AND METHODS: We determined the angiogenic properties of KS cell-secreted products and the HIV-1-tat gene product in vivo. Cell-free secreted products (KS-CM) from cultured epidemic and sporadic KS spindle cells or recombinant (r) HIV-1 tat protein were injected into mice with a matrix support (Matrigel). RESULTS: KS-CM produced lesions carrying all the phenotypic hallmarks of KS, as observed by light and electron microscopy: spindle-shaped cells, haemorrhages and an inflammatory infiltrate, as well as Factor VIII-positive endothelial cells lining new blood vessels. Electron microscopy indicated an initial granulocyte invasion, with spindle-cell migration and neocapillary formation in the centre of the matrix. These lesions required the cofactor heparin; KS-CM or heparin alone were poorly angiogenic. A less intense angiogenesis, with lower cellularity and few granulocytes, was observed in basic fibroblast growth factor (bFGF)/heparin lesions, indicating that factors other than bFGF are present in the KS spindle-cell products. When the collagenase inhibitor tissue inhibitor of metalloproteinases (TIMP)-2 was added to the sponges, KS-CM-induced angiogenesis was reduced by approximately 65% and bFGF-induced angiogenesis inhibited completely. Recombinant HIV-1 tat protein, a growth factor for KS cells, induced vascularization that was also enhanced by heparin, implying that HIV-1 tat could contribute to the aetiology of HIV-associated KS. CONCLUSIONS: KS-like lesions were obtained by injecting cell-free secreted products, suggesting that KS is a 'self-propagating' proliferative lesion caused by a cytokine imbalance and not a true neoplasm. Heparin-binding factors appear to be involved, and HIV-1 tat angiogenic properties implicate this molecule in AIDS-associated KS. Inhibition of KS-CM-induced KS-like lesions by TIMP-2 suggests that metalloproteinase inhibitors could be potential therapeutic agents for KS.

Monocyte-derived dendritic cells and monocytes migrate to HIV-Tat RGD and basic peptides.

OBJECTIVE AND DESIGN: Extracellular Tat released from HIV-1-infected cells is a mitogenic and motogenic factor for endothelial and Kaposi's sarcoma (KS)-derived cells and is angiogenic in vivo. Here we show for the first time that Tat induces migration of human dendritic cells in a concentration-dependent manner and that the Arg-Gly-Asp (RGD) and basic Tat peptides contribute to dendritic and monocyte cell migration. In vivo, Tat stimulates invasion of macrophages into a matrigel sponge. METHODS: Monocyte and dendritic cell chemotaxis was assessed using the Boyden chamber assay. RESULTS: Tat induced migration of monocyte-derived dendritic cells at the same levels as the N-formyl-Met-Leu-Phe peptide, and of monocytes at levels comparable to RANTES. Peptide mapping of the chemotactic activity of Tat showed that the RGD domain, which has been shown to support integrin-mediated cell migration, and the basic domain which binds and activates the tyrosine kinase receptor KDR on endothelial cells, both had activity. Antibody-blocking experiments indicate that responses to the RGD domain was inhibited by beta1 and alpha vbeta3 integrin blocking antibodies. Combination of the Tat RGD and basic peptides did not show additive effects; however, Tat co-operated with macrophage-chemotactic protein or RANTES in inducing monocyte migration. CONCLUSIONS: Our results show that Tat can act as a chemoattractant for dendritic cells, and that both the RGD and basic domains are involved in this response. These same domains attract monocytes. The alpha vbeta3 and beta1 integrins are equally involved in Tat-induced monocyte migration, while the alpha vbeta3 integrin largely mediates the dendritic cell response to Tat.

Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities.

Somatostatin was reported to inhibit Kaposi's sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.

HIV-Tat dependent chemotaxis and invasion, key aspects of tat mediated pathogenesis.

Extracellular Tat acts as a pleiotropic molecule inducing several biological effects on different target cells. Tat stimulates the chemotaxis of numerous cell types and it appears to have oncogenic activities, including acting as a co-factor for Kaposi's sarcoma. The Tat protein has been shown to bind integrins through an RGD amino acid motif. Tat is an angiogenic factor able to induce the migration and invasion of endothelial and KS cells through the interaction of its basic domain with the VEGF receptor VEGFR2 (Flk-1/KDR). We have also found that Tat is able to mimic chemokines, activating monocyte migration through the chemokine like' cysteine-core domain. Tat is a chemoattractant for dendritic cells, and both the RGD and basic domains appear to be involved in this response. In a recent study we demonstrated that Tat is chemotactic for PMN and induces Ca2+ mobilization in vitro. Experiments using synthetic peptides showed that Tat activities on PMN are mediated by the chemokine like' region. Finally Tat is also able to induce B cell chemotaxis, while its activity on helper T cells has not yet been clarified. Here we review data on Tat-dependent chemotaxis and discuss the possible implications in Tat mediated pathogenesis.

Tissue inhibitors of metalloproteases: regulation and biological activities.

A central role in tissue invasion is played by proteases that degrade extracellular matrices; in particular specific metalloproteases (MMPs) have been frequently correlated with the invasive potential of tumor cells and with the angiogenic process. MMPs are tightly regulated by molecules controlling their activation and by specific inhibitors of MMPs, known as the Tissue Inhibitors of MetalloProteases or TIMPs. Four TIMP family members are currently known. An imbalance between MMPs and TIMPs is linked to the degradation of the extracellular matrix associated with several physiologic and pathologic events including angiogenesis, invasion and metastasis. TIMPs are not only the 'guardians' of tissue degradation, they are able to control cell proliferation and cell survival as well. Given the critical role that TIMPs play, it is vital to know how the expression of TIMPs is controlled. Here we review the major biological properties and the molecular regulation of the TIMP expression.

The in vitro invasiveness and interactions with laminin of K-1735 melanoma cells. Evidence for different laminin-binding affinities in high and low metastatic variants.

The invasive and metastatic characteristics of cloned cells derived from the K-1735 murine melanoma were investigated. Cell lines which are highly metastatic in mice were found to be invasive in vitro, and to show an enhanced attachment to, spreading on and migration toward laminin. As attachment, spreading and directional migration are thought to be receptor-mediated events, the binding of laminin to these cells was studied. Biotinylated laminin was used to evaluate receptor binding by fluorescence activated cell sorting (FACS) and this method was compared with that in which the binding of radioactive laminin is measured. Both studies revealed that metastatic K-1735 cells (a) have more receptors for laminin compared with non-metastatic cells and (b) exhibit a second population of low-affinity binding sites not present on the non-metastatic cells. The differences in receptor number and type may account for the greater interaction of metastatic cells with laminin and their invasive phenotype.

Altered production of laminin and nidogen by high and low metastatic variants of murine melanoma cells.

An altered ability to interact with and degrade extracellular matrix molecules is a common feature of the malignant phenotype. Although changes in the expression of matrix proteins in metastases in vivo are relatively well documented, little is known about the changes in matrix production by malignant cells in culture. Here we have examined the synthesis of the basement membrane components laminin and nidogen (entactin) by low and high metastatic variants of the K-1735 murine melanoma cells. Protein deposition was examined by western blotting as well as immunofluorescence; protein synthesis was examined by immunoprecipitation with specific antibodies. Gene expression was also evaluated by measuring steady-state mRNA levels using cDNA probes on northern and dot-blots. Laminin gamma 1 levels appeared to be similar in both high and low metastatic lines; however, the high metastatic lines had reduced levels of the laminin beta 1 chain. On the contrary, nidogen expression was observed only in the high metastatic lines. Traces of a laminin alpha chain were present only in immunoprecipitates of the low metastatic cells and could not be detected in the high metastatic cells. Both high and low metastatic cells deposited an extracellular matrix of basement membrane components, with laminin deposition decreased in high metastatic cells. Modified expression, production, and deposition of basement membrane components in high metastatic melanoma cells could be involved in their altered interactions with the extracellular matrix.

Inhibition of angiogenesis by type I interferons in models of Kaposi's sarcoma.

Kaposi's Sarcoma (KS) is a pathology which occurs with increased frequency and in a particularly aggressive form in AIDS patients. The HIV-1 Tat protein appears to be an important co-factor in the induction of the extensive neo-vascularization associated with AIDS-KS. Tat acts as a chemoattractant for endothelial cells in vitro, inducing both chemotactic and invasive responses. Several clinical trials have been performed testing the effectiveness of diverse biological agents in therapy of KS, among these the type I interferons. Type I IFNs have diverse biological functions besides their anti-viral activity, including anti-angiogenic properties. We have shown that IFN alpha and IFN beta are potent inhibitors of both primary and immortalized endothelial cell migration and morphogenesis in vitro as well as neo-angiogenesis induced by HIV-1 Tat in vivo. The inhibitory effect of IFN class I on HIV-Tat associated angiogenesis further supports its use as a therapy for epidemic Kaposi's sarcoma. The use of recombinant IFNs at the levels required to obtain a therapeutic effect are associated with side effects and toxicity, therefore we are now developing a gene therapy approach for constant and local delivery type I IFNs.

Antiangiogenic activity of chemopreventive drugs.

Tumors growing within the host form dynamic aberrant tissue that consists of host components, including the stroma, an expanding vasculature and often chronic inflammation, in addition to the tumor cells themselves. These host components can contribute to, rather than limit, tumor expansion, whereas deprivation of vessel formation has the potential to confine tumors in small, clinically silent foci. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk, or of micrometastases after surgical removal of a primary tumor. Our analysis of potential cancer chemopreventive molecules including N-acetylcysteine, green tea flavonoids and 4-hydroxyphenyl-retinamide has identified antiangiogenic activities that could account--at least in part--for the tumor prevention effects observed with these compounds. These drugs appear to target common mechanisms of tumor angiogenesis that may permit identification of critical targets for antiangiogenic therapy and antiangiogenic chemoprevention.

The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis.

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.

Phenotypic alterations in Kaposi's sarcoma cells by antisense reduction of perlecan.

Metastasis is a sequence of events including proliferation, migration, adhesion, invasion and subsequent metastatic growth of tumour cells in distant organs. We previously showed that highly metastatic variants of murine melanoma cells express higher levels of the basement membrane proteoglycan perlecan than low or non metastatic variants and expression of an antisense perlecan can reduce metastatic potential. In contrast, antisense expression of perlecan in fibrosarcoma cells was reported to enhance tumorigenesis. To better understand the role of perlecan in angiogenesis we have transfected KS-IMM, an immortalized cell line derived from a human Kaposi s sarcoma, with an antisense perlecan construct and investigated the positive/negative role of perlecan in KS. KS-IMM cells were transfected with either empty vector (neo) or the antisense perlecan construct and clones were isolated. Immuno-blot analysis showed a reduction of perlecan levels in two (AP3 and AP4) isolated clones, in Northern blot analysis endogenous perlecan was undetectable in the AP3 and AP4 clones, while it was present in the neo control clones. AP clones had a reduced migration to HGF in Boyden chambers as compared to neo clones. Proliferation in low serum or serum-free conditions was strongly reduced in the AP clones as compared to the neo control cells. The neotransfected cells showed rapid proliferation in low serum supplemented with HGF and VEGF, while antisense transfected clones showed little response. Finally, AP-trasfected KS-IMM cells had significantly reduced migration to VEGF and HGF with respect to controls. In contrast, when the AP transfected cells were injected in nude mice they paradoxically showed enhanced tumor growth as compared to controls. Our preliminary data indicate that perlecan reduction plays a crucial role on Kaposi s sarcoma cell migration and proliferation in vitro. However, in vivo KS-IMM depleted of perlecan had a growth advantage. A possible hypothesis is that perlecan is necessary for growth of KS-IMM cells in vitro, however its down-regulation might promote angiogenesis through increased angiogenic growth factor diffusion, resulting in enhanced tumor growth in vivo.