Johan R. Westphal

Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis.

Vascular density in melanoma xenografts correlates with vascular permeability factor expression but not with metastatic potential.

We studied the relation between tumour vascular density and tumour growth rate, metastatic incidence and vascular permeability factor (VPF) mRNA levels in a human xenograft model described previously. Vascular density was determined by automated image analysis. Xenografts derived from cell lines BLM and MV3 showed the highest mean vascular density (MVD), the highest in vivo growth rate, high VPF mRNA levels and rapid development of lung metastases. Xenografts of cell lines M14, Mel57 and MV1 showed a significantly lower degree of vascularization, lower in vivo growth rates and lower levels of VPF mRNA, but formed lung metastases with a similar incidence as those of BLM and MV3. Xenografts from cell line 1F6 did not form lung metastases, whereas tumours derived from a spontaneous mutant of 1F6, designated 1F6m, gave rise to lung metastases to the same extent as Mel57, M14 and MV1 tumours. MVD values in 1F6 and 1F6m xenografts, VPF mRNA levels and in vivo growth rates of 1F6 and 1 F6m xenografts, however, were similar. In conclusion, in the melanoma xenograft model vascular density is correlated with in vivo growth rate and with in vitro VPF mRNA levels, but not with the ability to metastasize.

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators.

Angiostatin is a tumor‐derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin‐generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western‐blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder‐carcinoma and 6 out of 7 prostate‐carcinoma cell lines showed intermediate to potent angiostatin‐generating activity. In contrast, only 2 out of 7 colon‐carcinoma and 2 out of 9 renal‐cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell‐line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI‐1 antigen in the conditioned media, and correlated the results with angiostatin‐generating capacity. Whereas prostate‐ and bladder‐carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non‐producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin. Int. J. Cancer 86:760–767, 2000.

An improved procedure to quantify tumour vascularity using true colour image analysis. Comparison with the manual hot-spot procedure in a human melanoma xenograft model

In a number of recent papers, the degree of tumour vascularization has been described as a promising new prognostic factor. Methods for the assessment of vascular density involve immunohistochemical staining of the vasculature, followed by counting the number of vessel profiles in the angiogenic hot spot. One of the problems of this procedure is the selection of the angiogenic hot spot, which has been described as being subject to inter‐observer variation. In this study, the value of true colour image analysis in reducing inter‐observer variation has been assessed. Highly (MV3) and poorly (M14) vascularized human melanoma xenografts were used to evaluate the image analysis procedure, and the image analysis results were compared with results from the conventional manual hot‐spot procedure. Assessment by image analysis was performed on measurement fields covering the entire tumour tissue specimens rather than on a single hot‐spot field. Also, by selecting the most densely vascularized area from all fields assessed by the semi‐automatic procedure, it was possible to objectify the hot spot selection (automated hot‐spot procedure). Manual assessment showed a good correlation between two independent observers for MV3 xenografts (r‐0·74, P‐0·014), but a poor correlation for M14 xenographs (r‐0·4, P>0·05). Automated assessment by different operators showed good correlations for both MV3 xenografts (r‐0·99, P<0·001) and M14 xenografts (r‐0·80, P‐0·006). It is concluded that although both manual vessel counting and semi‐automated image analysis can differentiate between the level of vascularization in the two types of xenograft (P<0·001 for both methods), the automated method is favourable in that it showed no significant inter‐observer effects. In M14 xenografts, the manual hot‐spot vessel densities did not correlate well with the automated hot‐spot densities (r‐0·27, P>0·05), indicating that selection of angiogenic hot spots in this tumour type is indeed subject to observer bias. The automated hot‐spot vessel densities were a reliable indicator of overall tumour vessel density in both tumour types. Image analysis allows analysis of vessel subclasses based on morphological criteria such as vessel profile area or diameter. In the model system used, the discrimination between MV3 and M14 xenografts was further enhanced by selectively examining vessels with diameters between 6 and 9 μm (P<0·0005). In conclusion, image analysis appears to offer an objective and more reproducible method to quantify tumour vascularity than manual counting of vessel profiles in the hot spot. Analysis of subclasses of vessels may further enhance the value of vessel density measurements in discriminating between tumour types differing in biological behaviour.

Expression of endoglin in psoriatic involved and uninvolved skin

Endoglin is a glycoprotein with TGF-beta binding capacity and is predominantly expressed on endothelial cells. In psoriasis, TGF-beta has appeared to play a role in the extravasation of peripheral blood mononuclear cells via the endothelium. In order to find out more about the role of endoglin in psoriasis, immunohistochemical staining with PN-E2, a novel anti-endoglin, and of PAL-E, recognizing vascular endothelium, was carried out in psoriatic involved, psoriatic uninvolved and normal skin. The expression of the antigens was assessed semi-quantitatively using a five-point scale. In psoriatic involved skin, a high endoglin expression was found. In psoriatic uninvolved skin, however, we found that endoglin expression was significantly decreased compared with normal skin. The relevance of these findings to the pathogenesis of psoriasis is discussed.

Anti-angiogenic treatment of human cancer pitfalls and promises

The dependence of solid tumors on angiogenesis for sustained growth and the formation of distant metastases is now an established concept in tumor biology (Folkman, 1990; Fidler and Ellis, 1994). When this concept was still in its infancy, the potential of inhibiting angiogenesis as a novel anti-tumor treatment was already recognized (Folkman, 1971). From a theoretical point of view, the anti-angiogenic approach has a number of advantages over conventional anti-cancer treatments. First, in many solid tumors, high interstitial pressure blocks the entry of chemotherapeutic agents into the tumor cell compartment. Anti-angiogenic compounds, on the other hand, target the endothelial cells facing the vessel lumen. Interstitial pressure is, therefore, less a barrier for these agents (Jain, 1987). Second, therapy resistance based on genetic instability is unlikely to develop (Kerbel, 1997). Finally, as angiogenesis does not occur in the adult organism under physiologic circumstances (with the exceptions of wound healing and certain stages of the female reproductive cycle), unwanted side effects of anti-angiogenic therapy are not to be expected, provided that the therapy is sufficiently specific. During the last decade, many anti-angiogenic therapies have been tested. Three main types of approaches can be discerned (summarized in Fig. 1). Most straightforward are attempts to attack the existing tumor vasculature directly with toxins, or procoagulant molecules for example. Other agents interfering with specific steps of the angiogenic process have been identified, thereby inhibiting the development of new vessels. Finally, the discovery of tumorderived anti-angiogenic substances has led to extensive research efforts to characterize, adapt, and produce these molecules for clinical use. Several excellent reviews have dealt with the detailed characteristics and possible applications of each group of molecules (Folkman, 1995a,b; Folkman, 1996; Klagsbrun, 1999). In this commentary, we shall briefly evaluate such anti-angiogenic strategies, and present suggestions for possible future approaches.

The proliferative response of human T cells to allogeneic IFN-γ-treated endothelial cells is mediated via both CD2/LFA-3 and LFA-1/ICAM-1 and -2 adhesion pathways

We studied the proliferative response of purified human peripheral blood T lymphocytes (contaminated with less than 0.1% monocytes) to allogeneic MHC class II molecules expressed by endothelial cells (EC) or fibroblasts (FB). In vitro expression of MHC class II molecules was induced by gamma-interferon (IFN-gamma) treatment. The MHC class II expression levels after IFN-gamma treatment on both cell types were comparable. No T cell proliferation was found in the presence of either untreated or IFN-gamma-treated FB, and a marginal proliferation in the presence of untreated EC. IFN-gamma-treated EC, however, were able to induce significant T cell growth. The previously established role of MHC class II molecules in allogeneic T cell proliferation was confirmed in inhibition experiments with monoclonal antibody (mAb) against MHC class II or CD4. In this model, we tested the involvement of a number of adhesion molecules by adding mAbs to cocultures of T cells and IFN-gamma-treated EC. Monoclonal antibodies directed against CD31, CD26, B7/BB1, E-selectin, CD44, VLA-4 alpha-chain and VCAM-1 had no effect, whereas moderate inhibition was observed with anti-VLA-beta-chain and anti-LFA-3. A distinct inhibition of T cell proliferation was observed with mAbs directed against LFA-1, CD2, or a combination of anti-ICAM-1 and -2. Combinations of mAbs directed against T cell adhesion molecules (LFA-1, CD2, VLA-4) or EC adhesion molecules (ICAM-1, and -2, LFA-3, VCAM-1) were able to block T cell proliferation for 100 and 80% respectively. We conclude that CD2/LFA-3 and LFA-1/ICAM interactions are crucially involved in allogeneic T cell/EC interactions.

The role of adhesion molecules in endothelial cell accessory function

Abbreviations: EC endothelial cells, EGF epidermal growth factor, ELAM endothelial cell leukocyte adhesion molecule, HEV high endothelial venule, HUVEC human umbilical vein endothelial cell, ICAM inter-cellular adhesion molecule, IL interleukin, LFA leukocyte-function associated antigen, M H C major histocompatibility complex, NCAM neural cell adhesion molecule, PAF platelet activating factor, SCR short consensus repeat, VCAM vascular cell adhesion molecule, VLA very late antigen

Plasminogen activators are involved in angiostatin generation in vivo in benign and malignant ovarian tumor cyst fluids.

In many tumor types, angiogenesis is the net result of pro- and anti-angiogenic mediators and correlated with metabolic activity, growth, and degree of malignancy. One of the first discovered anti-angiogenic compounds is angiostatin, a proteolytic fragment of plasminogen. The requirements for in vivo angiostatin generation have not yet been determined. We investigated the levels of plasminogen and angiostatin by western blotting and of components of the plasminogen activator complex by ELISA in cyst fluid derived from benign and malignant ovarian tumors. Fluid samples from functional ovarian follicles, dermoid cysts and endometriotic lesions were evaluated separately. When no or minimal amounts of plasminogen were present in the cyst fluids, angiostatin was generally absent as well, irrespective of plasminogen activator concentrations. When plasminogen was present, the degree of conversion of plasminogen to angiostatin was significantly correlated with the level of uPA, and, to a lesser extent, to the tPA level. However, angiostatin was also found in a number of cyst fluid samples with minimal or no plasminogen activators, suggesting the involvement of other angiostatin generating proteases in these samples. Conversely, no angiostatin was observed in a number of cyst fluid samples containing both plasminogen and plasminogen activators. The presence of an inhibitor of the enzymatic activity of uPA and/or tPA, like PAI-1, may explain this finding. Our data show that plasminogen activators are clearly involved in in vivo angiostatin formation in ovarian cysts. Most likely, however, other proteases, as well as inhibitors of plasminogen activators, are involved as well.

The adhesion of T lymphocytes to human endothelial cells and brain pericytes is differentially regulated

Introduction: In Alzheimers disease (AD) patients, the complement components Clq, CA and C3 can be detected in amyloid (B/A4) plaques, one of the hallmarks of AD. In AD the formation of the lyric membrane attack complex (MAC) is supposed to be an Intermediate between the B/A4 depos/ts and the obu~vod neuroto0dcity. Matadals mid Metim~: We analyzed In an extensive histochemtcul study the presence of a number of complement components and regulatory proteins in AD temporal corte~ Clq, CA and C3 activation products, but not Clr, Cls and C1esterase inhibitor were colonallzed with B/A4 depmits. In addition, no late components C5, C7, C9, nor MAC and its inhibitor CD59 could be detected in ~/A4 positive plaques. On the other hand, the MAC inltibitors clnsterin and vltronectln are present in both classical and diffuse plaques. CoadW[oa: The al~ence of the cytolyflc C5b-9 complex in AD brains suggests, that in AD complement does not function as the proposed in0ammtoiy medlator betwe¢~ B/A4 deposits and nettroflbrlllary changes. Probebly, in the brain the locally produced Clq and also C3 and the m u l ~ u a l Inhibitors cinstetin and viU-one~da serve another function than initiation or regulation of complement activation.