Patricia Ropraz

Phenotypic Heterogeneity of Rat Arterial Smooth Muscle Cell Clones Implications for the Development of Experimental Intimal Thickening

It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro.

Heterogeneity of Smooth Muscle Cell Populations Cultured From Pig Coronary Artery

Objective — Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. Methods and Results — By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a “hill-and-valley” growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-β2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. Conclusions — Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Rat aortic smooth muscle cells isolated from different layers and at different times after endothelial denudation show distinct biological features in vitro.

Endothelial denudation by balloon injury of the rat aorta induces the development of a neointima as a consequence of the migration and proliferation of smooth muscle cells (SMCs). Initially, intimal SMCs show a dedifferentiated phenotype, which reverts to a normal differentiated phenotype after endothelial cells have resurfaced the vessel lumen. We investigated in vitro the proliferative and phenotypic features of SMCs from different layers of rat aorta isolated 15 and 60 days after endothelial denudation. Freshly isolated intimal cells 15 days after balloon injury (IT-15) appeared rounded and showed a decreased content of alpha-smooth muscle actin, smooth muscle myosin, and desmin compared with intimal cells isolated 60 days after balloon injury (IT-60). No morphological and cytoskeletal differences were observed among freshly isolated IT-60 cells and other medial populations, which included medial SMCs that underlie the intimal thickening. In culture, IT-15 cells showed increased proliferative activity both in monolayers and in free-floating collagen lattices. Decreased expression of alpha-smooth muscle actin and smooth muscle myosin was documented in IT-15 cells compared with IT-60 cells and other medial SMC populations in monolayer. Moreover, IT-15 cells suspended in collagen lattices were poor at contracting these collagen lattices compared with IT-60 and control SMCs. IT-60 cells were equivalent to control SMCs at lattice contraction except for a temporary delay at day 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Age influences the replicative activity and the differentiation features of cultured rat aortic smooth muscle cell populations and clones.

The replicative activity and the differentiation features of aortic smooth muscle cells (SMCs) cultured as whole populations or clones from newborn (4-day-old), young adult (6-week-old), and old (18-month-old) rats were studied by means of cell counting, [3H]thymidine incorporation, and measurement of the expression of cytoskeletal proteins and mRNAs. In whole populations at the fifth passage, replicative activity increased and differentiation features (ie, expression of alpha-smooth muscle actin, desmin, and smooth muscle myosin heavy chains) decreased with increasing age of the donor animal. SMC clones derived from newborn or young adult rats showed more differentiated cytoskeletal features than their parental populations; however, most SMC clones from old rats showed dedifferentiated features similar to those observed in their parental populations. Our results suggest that (1) SMCs of the rat aortic media behave as a heterogeneous population; (2) cultured whole SMC populations behave differently from clones as far as their replicative activity and differentiation features are concerned; and (3) SMCs derived from old rats, whether grown as whole populations or as clones, dedifferentiate more substantially and replicate more actively than corresponding cultures from newborn or young adult rats when submitted to the same amount of serum growth factors; these differences may play a role in arterial development as well as in the formation and evolution of the atheromatous plaque.

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

High levels of cellular retinol binding protein-1 expression in leiomyosarcoma: possible implications for diagnostic evaluation

Abstract. Retinoid bioavailability is regulated by the activity of specific cytoplasmic receptors. High levels of cellular retinol binding protein-1 (CRBP-1) have been documented during experimental arterial and wound-healing processes, but data concerning neoplastic smooth muscle tissues are scarce and/or controversial. This study reports that the expression of CRBP-1 is markedly higher in uterine and gastrointestinal leiomyosarcomas than in leiomyomas and normal myometrium; CRBP-1 was practically absent in normal gastrointestinal smooth muscle tissue. CRBP-1 positivity was particularly elevated in the epithelioid variant of leiomyosarcoma; it was associated with increased proliferative and apoptotic rates and inversely related to smooth muscle differentiation evaluated by α- and γ-smooth muscle actin and desmin expression. Western blotting and reverse-transcription polymerase chain reaction confirmed the observations concerning CRBP-1 and actin isoform expression and revealed higher NF-κ-Bp65 and RARα and lower Bax protein levels in leiomyosarcoma than in the other conditions. These findings document that a high CRBP-1 expression is associated with smooth muscle malignancy and suggest that CRBP-1 expression represents a new useful marker for the classification of unusual variants of smooth muscle tumors.

Junctional adhesion molecule B interferes with angiogenic VEGF/VEGFR2 signaling

De novo formation of blood vessels is a pivotal mechanism during cancer development. During the past few years, antiangiogenic drugs have been developed to target tumor vasculature. However, because of limitations and adverse effects observed with current therapies, there is a strong need for alternative antiangiogenic strategies. Using specific anti‐junctional adhesion molecule (JAM)‐B antibodies and Jam‐b‐deficient mice, we studied the role in antiangiogenesis of JAM‐B. We found that antibodies against murine JAM‐B, an endothelium‐specific adhesion molecule, inhibited microvessel outgrowth from ex vivo aortic rings and in vitro endothelial network formation. In addition, anti‐JAM‐B antibodies blocked VEGF signaling, an essential pathway for angiogenesis. Moreover, increased aortic ring branching was observed in aortas isolated from Jam‐b‐deficient animals, suggesting that JAM‐B negatively regulates proangiogenic pathways. In mice, JAM‐B expression was detected in de novo‐formed blood vessels of tumors, but anti‐JAM‐B antibodies unexpectedly did not reduce tumor growth. Accordingly, JAM‐B deficiency in vivo had no impact on blood vessel formation, suggesting that targeting JAM‐B in vivo may be offset by other proangiogenic mechanisms. In conclusion, despite the promising effects observed in vitro, targeting JAM‐B during tumor progression seems to be inefficient as a stand‐alone antiangiogenesis therapy.—Meguenani, M., Miljkovic‐Licina, M., Fagiani, E., Ropraz, P., Hammel, P., Aurrand‐Lions, M., Adams, R. H., Christofori, G., Imhof, B. A., Garrido‐Urbani, S. Junctional adhesion molecule B interferes with angiogenic VEGF/VEGFR2 signaling. FASEB J. 29, 3411‐3425 (2015). www.fasebj.org

Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours

Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14dimCD16+ patrolling and CD14+CD16+ intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.Circulating myeloid cells can leave the vasculature to infiltrate tumours and are thought to contribute to tumour angiogenesis. Here the authors live image monocytes that migrate to xenograft tumours and map an extravasation cascade of human proangiogenic monocytes into the tumour.

Junctional adhesion molecule C (JAM-C) dimerization aids cancer cell migration and metastasis

Most cancer deaths result from metastasis, which is the dissemination of cells from a primary tumor to distant organs. Metastasis involves changes to molecules that are essential for tumor cell adhesion to the extracellular matrix and to endothelial cells. Junctional Adhesion Molecule C (JAM-C) localizes at intercellular junctions as homodimers or more affine heterodimers with JAM-B. We previously showed that the homodimerization site (E66) in JAM-C is also involved in JAM-B binding. Here we show that neoexpression of JAM-C in a JAM-C-negative carcinoma cell line induced loss of adhesive property and pro-metastatic capacities. We also identify two critical structural sites (E66 and K68) for JAM-C/JAM-B interaction by directed mutagenesis of JAM-C and studied their implication on tumor cell behavior. JAM-C mutants did not bind to JAM-B or localize correctly to junctions. Moreover, mutated JAM-C proteins increased adhesion and reduced proliferation and migration of lung carcinoma cell lines. Carcinoma cells expressing mutant JAM-C grew slower than with JAM-C WT and were not able to establish metastatic lung nodules in mice. Overall these data demonstrate that the dimerization sites E66-K68 of JAM-C affected cell adhesion, polarization and migration and are essential for tumor cell metastasis.

The pyrazolyl-urea GeGe3 inhibits tumor angiogenesis and reveals dystrophia myotonica protein kinase (DMPK)1 as a novel angiogenesis target

The limitation of targeting VEGF/VEGFR2 signalling to stop angiogenesis in cancer therapy has been blamed on re-activation of alternative receptor tyrosine kinases by compensatory angiogenic factors. Targeting MAPK and PI3K signaling pathways in endothelial cells may be an alternative or complementary approach. Herein we aimed to evaluate the antitumor and antiangiogenic potential of a novel pyrazolyl-urea kinase inhibitor, GeGe3, and to identify its kinase targets. We found GeGe3 to inhibit the proliferation of HUVEC and endothelial tube formation. GeGe3 impaired inter-segmental angiogenesis during development of zebrafish embryos. In mice, GeGe3 blocked angiogenesis and tumor growth in transplanted subcutaneous Lewis Lung Carcinomas. Screening for GeGe3-targeted kinases revealed Aurora B, Aurora C, NEK10, polo-like kinase (PLK)2, PLK3, DMPK1 and CAMK1 as candidate targets. Biochemical analysis of these kinases showed DMPK1 regulation upon VEGF challenge. Investigation of the role of DMPK1 in endothelial cells revealed DMPK1 as a novel mediator of angiogenesis that controls the activation of MAPK signaling, proliferation and migration. GeGe3 alters angiogenesis by targeting DMPK in tumor endothelial cells and pericytes. The pyrazolyl-urea GeGe3, a novel blocker of MAPK and PI3K pathways, strongly inhibits physiological and tumor angiogenesis. We also report GeGe3-targeted kinase DMPK as a novel mediator of angiogenesis.