Nicolae Mirancea

Angiogenesis Inhibition by Vascular Endothelial Growth Factor Receptor-2 Blockade Reduces Stromal Matrix Metalloproteinase Expression, Normalizes Stromal Tissue, and Reverts Epithelial Tumor Phenotype in Surface Heterotransplants

Inhibition of vascular endothelial growth factor (VEGF) signaling, a key regulator of tumor angiogenesis, through blockade of VEGF receptor (VEGFR)-2 by the monoclonal antibody DC101 inhibits angiogenesis, tumor growth, and invasion. In a surface xenotransplant assay on nude mice using a high-grade malignant squamous cell carcinoma cell line (A-5RT3), we show that DC101 causes vessel regression and normalization as well as stromal maturation resulting in a reversion to a noninvasive tumor phenotype. Vessel regression is followed by down-regulation of expression of both VEGFR-2 and VEGFR-1 on endothelial cells and increased association of alpha-smooth muscle actin-positive cells with small vessels indicating their normalization, which was further supported by a regular ultrastructure. The phenotypic regression of an invasive carcinoma to a well-demarcated dysplastic squamous epithelium is accentuated by the establishment of a clearly structured epithelial basement membrane and the accumulation of collagen bundles in the stabilized connective tissue. This normalization of the tumor-stroma border coincided with down-regulated expression of the stromal matrix metalloproteinases 9 and 13, which supposedly resulted in attenuated turnover of extracellular matrix components permitting their structural organization. Thus, in this mouse model of a human squamous cell carcinoma cell line, blockade of VEGF signaling resulted in the reversion of the epithelial tumor phenotype through stromal normalization, further substantiating the crucial role of stromal microenvironment in regulating the tumor phenotype.

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermo- epidermal junction in organotypic co-culture

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.

Basement Membranes in Skin Are Differently Affected by Lack of Nidogen 1 and 2

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.

Rapid vessel regression, protease inhibition, and stromal normalization upon short-term vascular endothelial growth factor receptor 2 inhibition in skin carcinoma heterotransplants.

Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and blockade of VEGF receptor 2 (VEGFR-2), with the monoclonal antibody DC101, inhibits angiogenesis and tumor growth. To examine the short-term effects of DC101, we surface transplanted the squamous cell carcinoma cell line A5-RT3 onto nude mice. After short-term treatment with DC101, we observed rapid reduction in vascularization and reversion of the tumor phenotype. Beginning 24 hours after treatment, VEGFR-2 inhibition resulted in decreased vessel density within the tenascin-c-staining tumor-associated stroma and reduced endothelial cell proliferation. Stromal expression of matrix metalloproteinase-9 and -13 was drastically reduced 96 hours after VEGFR-2 inhibition as detected by in situ hybridization and in situ zymography. Moreover, the morphology of the tumor-stroma border changed from a highly invasive carcinoma to a well-demarcated, premalignant phenotype. The latter was characterized by the appearance of a regular basement membrane in immunostaining and ultrastructural analyses. These findings suggest that VEGFR-2 inhibition by DC101 evokes very rapid reduction of preformed vessels and decreases both stromal protease expression and gelatinolytic activity, resulting in the modulation of the tumor-stroma border zone and reversion of the tumor phenotype. Thus, short-term inhibition of VEGF signaling results in complex stromal alterations with crucial consequences for the tumor phenotype.

Control of basement membrane formation in skin-organotypic 3d-coculture

Basement membrane (BM) formation was functionally dissected in 3d-cocultures of human keratinocytes (HK) and fibroblasts (human/mouse, HF/MFf) by either blocking interactions or implementing molecular deficiencies. This was supposed to complement knockout mouse studies, where loss or functional defects of collagen-IV, laminins, nidogen, or perlecan are causing embryonic or neonatal death. HK or HaCaT cells were grown on collagen gels harboring hf or mf from normal or ko-mice. To block nidogen-binding to laminin-10 the corresponding laminin-fragment (gamma1-iii3-5, L-gamma-f) was applied. BM-formation was surveyed by immunofluorescence (IF), regular (EM) and immuno-electron microscopy (IEM). In 3d-cocultures of HK and HF L-gamma-f blocked deposition of nidogen, laminin-10, and perlecan, while collagen-IV appeared normal. Although the hemidesmosome components laminin-5, BP180, and integrin alpha6beta4 were only mildly affected, EM and IEM revealed complete absence of BM, hemidesmosomes, and basal insertion of keratin filaments. To eliminate nidogen, made by fibroblasts, MF from nidogen1/nidogen2 ko-mice or crossbreds were employed. In 3d-cocultures with HaCaT cells nidogen1/2 (??/++)-MF abolished nidogen1-staining, but (??/+?)-mf reduced also largely nidogen2, collagen-IV, and drastically laminin-10. Total absence of nidogen (??/??) also deleted collagen-IV & laminin-5, integrins e.g. alpha6beta4 appearing still normal (IF). BM-formation could be entirely rescued by applying recombinant nidogens. In skin, perlecan can be apparently synthesized by both keratinocytes & fibroblasts. Accordingly, deficiency in either cell type did not affect BM-formation, demonstrated by combining either perlecan (?/?)-mf or HaCaT anti-sense-perlecan cells with respective normal partner cells. Thus, in this skin model BM-components are efficiently transported to their actual assembly site.