Telocytes inside of the peripheral nervous system - a 3D endoneurial network and putative role in cell communication.

In this paper, we developed the hypothesis concerning the reasons to assimilate endoneurial fibroblast-like dendritic phenotype [shortly termed endoneurial dendritic cells (EDCs)] to the endoneurial telocytes (TCs). We reviewed the literature concerning EDCs status and report our observations on ultrastructure and some immune electron microscopic aspects of the cutaneous peripheral nerves. Our data demonstrate that EDCs long time considered as fibroblasts or fibroblast-like, with an ovoidal nucleus and one or more moniliform cell extensions [telopodes (Tps)], which perform homocellular junctions, also able to shed extracellular microvesicles can be assimilated to TC phenotype. Sometimes, small profiles of basement membrane accompany to some extent Tps. Altogether data resulted from scientific literature and our results strength the conclusion EDCs are really TCs inside of the peripheral nervous system. The inner three-dimensional (3D) network of endoneurial TCs by their homo- and heterocellular communications appears as a genuine cell-to-cell communication system inside of each peripheral nerve.

Analysis of TP53 gene and particular infrastructural alterations in invasive ductal mammary carcinoma.

This study was conducted in order to determine the mutational status of TP53 gene and to determine some particular aspects from ultrastructural level in invasive mammary ductal carcinoma. The cellular signaling pathway involving the TP53 gene acts in biological deoxyribonucleic acid (DNA) repair processes and cell cycle arrest following a signal transmitted to the p53 protein when posttranslational changes occur in the cell due to stress induced in the cell by both intrinsic and extrinsic factors. Cellular stress activates the transcription factor function of the protein that initiates, as the case may be, either DNA repair or programmed cell death (apoptosis). The TP53 gene is commonly mutated in many human cancers and also has a highly polymorphic grade. To determine the mutational status of the exons 4-9 of the TP53 gene, we used extracted DNA from fresh breast tissue, and we analyzed it through direct sequencing. In mammary carcinoma, the mutation frequency of TP53 is running between 20-40% and, in regards the polymorphism, at least 14 different forms were identified, that are associated with cancer risk. The mutation type distribution showed a predominance of deletions and a reduced frequency of substitutions comparing with International Agency for Research on Cancer (IARC) database. Taken in consideration the importance of the tumor associated stroma in tumor development, we have also investigated some particular aspects at the infrastructural level of invasive mammary ductal carcinoma, notably concerning telocytes as tumor stroma interstitial cells by transmission electron microscopy analysis.

Molecular analysis of BRCA1 and BRCA2 genes by next generation sequencing and ultrastructural aspects of breast tumor tissue.

In this paper, we focus our interest on the dynamics alterations of the tumor-stroma interface at the ultrastructural level and to detect BRCA1 and BRCA2 mutations using next generation sequencing (NGS) of breast tumor tissue. Electron microscopic investigation revealed some peculiar infrastructural alterations of the tumor cells per se as well as of the tumor-stroma interface: invadopodia, shedding microvesicles, altered morphology and reduced number of telocytes, different abnormalities of the microvasculature. Tumor suppressor genes BRCA1 and BRCA2 are the genes with most hereditary predisposition to breast and ovarian cancer. An early identification of mutation within these genes is essential for determining classification and therapeutic approach to patients. Genetic tests used to determine mutations in BRCA1 and BRCA2 genes are laborious analysis methods which include, among others, NGS. We analyzed a total of eight samples, in which genomic DNA was amplified using Ion AmpliSeq panel BRCA1 and BRCA2. DNA libraries were created, amplified and sequenced with Ion Torrent Personal Genome Machine. The bio-information data obtained allow us to detect all known pathogenic mutation and uncertain polymorphisms.

Telocytes of the human adult trigeminal ganglion.

Telocytes (TCs) are typically defined as cells with telopodes by their ultrastructural features. Their presence was reported in various organs, however little is known about their presence in human trigeminal ganglion. To address this issue, samples of trigeminal ganglia were tested by immunocytochemistry for CD34 and examined by transmission electron microscopy (TEM). We found that TCs are CD34 positive and form networks within the ganglion in close vicinity to microvessels and nerve fibers around the neuronal-glial units (NGUs). TEM examination confirmed the existence of spindle-shaped and bipolar TCs with one or two telopodes measuring between 15 to 53 µm. We propose that TCs are cells with stemness capacity which might contribute in regeneration and repair processes by: modulation of the stem cell activity or by acting as progenitors of other cells present in the normal tissue. In addition, further studies are needed to establish if they might influence the neuronal circuits.

Telocyte - a particular cell phenotype. Infrastructure, relationships and putative functions.

Here we review on the infrastructure, relationships and putative role of a new cell phenotype termed telocyte. Ultrastructural and immunochemical characteristics of telocytes, their identification in different tissues, homo- and heterocellular telocyte's contacts and considerations concerning their putative role in normal as well as pathological conditions are largely presented. A special section of the paper is devoted to our opinion÷hypothesis concerning the possibility that, to some extent, endoneurial fibroblast dendritic phenotype (existed in the peripheral nerve endoneurium) can be assimilated to the telocyte or, at least to the telocyte-like. In this respect, we report about a body of evidence that endoneurial fibroblasts dendritic cells share some infrastructural characteristics with telocyte phenotype. Telocytes involvement in pathology, tissue engineering and regenerative medicine is also debated.

Relevant infrastructural alterations in invasive pancreatic ductal adenocarcinoma.

In this study, we focus our interest on some peculiar infrastructural abnormalities detected in a pancreatic cancer case. Our electron microscopic observations underline the high plasticity of the pancreatic parenchyma cells. Tumor pancreatic exocrine lesions are represented by putative ductal and acinar cells, which proliferate and grow in a haphazard pattern, detrimental to endocrine counterpart. The tumor cells do not exhibit neither a pure ductular or ductal nor a pure acinar phenotype, but tumor lesions represented by neoplastic ductal cells with invasive growth are by far prevalently. In our pancreatic cancer case, electron microscopic investigation clearly shows that a plethora of the epithelial cells from the tumor lesions contain large areas of autophagy leading to the pleomorphic inclusions represented by fibrillary÷filamentous inclusions frequently associated with hyaline-amorphous material, and secondary lysosomes. One of the mostly striking and important finding in this report for a case of pancreatic cancer is the high fragility (extensive dissolutions) of plasma membrane of tumor cells leading to pseudo-syncytia formation. Desmosomal junctions are severely altered, almost missing. Plasma membranes showed shedding membrane vesicles. Extravasated inflammatory cells contribute to the dramatic and extensive destructive areas of epithelial cells as well as tumor-stroma counterpart, including the basement membrane. All above severe infrastructural abnormalities, especially down regulation of cell-cell and cell-extracellular matrix adhesions might result in aberrant cell behavior and, consequently, much care should be taken for the postoperatory patient evolution.

Particular molecular and ultrastructural aspects in invasive mammary carcinoma.

Electron microscopic investigations of invasive mammary carcinoma tumors revealed that intercellular junctions, namely desmosomes are severely altered; some desmosomes became internalized. Tumor cells, especially by their invadopodia, generate and disseminate membrane vesicles, including exosomes, inside of peritumoral stroma. Telocytes, a new described interstitial/stromal cell phenotype, considered to play important roles in cell signaling, exhibited a reduced number of hetero-cellular contacts, which suggests a possible perturbation of tissular homeostasis modulation. Signaling PIK3/Akt pathway plays an important role both in carcinogenesis and in proliferation, differentiation, and cell survival. Alteration of this pathway has been observed in many human cancers, often involving an increase in the activity of PIK3CA, p110α catalytic subunit of PI3K. Our study confirms the high prevalence of PIK3CA mutations in breast cancer. In accordance with the results of the largest previous studies, 87.5% of mutations detected by DNA direct sequencing were hot spot mutations, most of them located in the kinase domain. High percentage of mutations detected by high-resolution melting makes the assay an attractive choice for mutation scanning, especially, in samples with low percentage of tumor cell.

Relevant infrastructural alterations in a pancreatic neuroendocrine tumor: an insulinoma case.

In this study, we focus our interest on some peculiar infrastructural abnormalities detected in an insulinoma case. Tumor pancreatic endocrine cells proliferated detrimental to exocrine counterpart, so that extensive areas of prevalent ß-tumor cells can be seen. Two phenotypes of ß-tumor cells can be identified: (1) ß-tumor cells with full euchromatic and nucleolated nuclei and (2) ß-tumor cells with heterochromatic and shrink nuclei. Because of stroma alteration, including basement membrane, cell-extracellular matrix junctions are also compromised. The mostly striking and important finding in this report for a case of insulinoma is the high fragility of plasma membrane of both two phenotypes of ß-tumor cells. Cell-cell junctions, especially desmosomal junctions are severely altered, almost missing, plasma membranes showed shedding membrane vesicles and extensive dissolutions leading to pseudo-syncytia formation. Extravasated blood cells, including inflammatory cells contribute to the dramatic and extensive destructive areas of epithelial cells as well as stroma counterpart. Moreover, also the inner cell cytomembranes exhibit abnormalities: many ß-tumor cells have excessive dilatations of nuclear envelope and endoplasmic reticulum. All above severe infrastructural abnormalities, especially down regulation of cell-cell and cell-extracellular matrix adhesions and plasma membranes fragility might result in aberrant cell behavior and, consequently, much care should be taken for the postoperatory patient evolution.

Early onset of podocytes apoptosis - a TEM study in streptozotocin-induced diabetic rats.

Cell death types are usually defined by morphological criteria. Even though podocyte loss is associated with various cell death mechanisms, podocyte apoptosis is rarely detected. The purpose of this study is to evaluate whether morphological signs of apoptotic cell death could be detected in early streptozotocin-induced diabetes in rats kidneys. There were used five Wistar rats, and renal tissue samples were drawn after three weeks of disease and further evaluated in transmission electron microscopy (TEM). Podocytes damage was indicated by two major findings: foot processes effacement, viewed as loss of cell processes, and chromatin condensation and margination (partial karyopyknosis: peculiar nuclear morphologies - partly normal, euchromatic, and partly positive for karyopyknosis and nuclear shrinkage). Mitotic glomerular endotheliocytes were also encountered. Podocytes cell death commitment and detachment appeared as concomitant events. However, karyopyknosis is not a specific feature of apoptosis. Thus, further biochemical evaluations are needed to distinguish between different pathways of podocytes death.

Basement membranes in skin: unique matrix structures with diverse functions?

The view of extracellular matrix (ECM) has evolved from a merely scaffolding and space filling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident micro-vasculature. The role of the non-polymeric components perlecan and particularly nidogen is exemplified by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deficiency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and 'minor' tissue-specific modifier or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements 'being essential for BM/or not' should be consequently referred to the developmental, tissue, and functional (e.g., repair) context.

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.

Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents.

In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability - over 12 weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4)cells/cm(2) on epidermal tissue quality equalled that of the standard density (2 x 10(5)cells/cm(2)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte-fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.

Reversion of tumor phenotype in surface transplants of skin SCC cells by scaffold-induced stroma modulation.

Interactions between cancer cells and the tissue microenvironment play an essential role in controlling tumor development and progression. Here, we report that stromal modulation induced by a biodegradable meshwork (Hyalograft 3D) inhibited tumor vascularization and invasion of the locally invasive low-grade malignant human HaCaT-ras II-4 keratinocytes in a surface xenotransplantation assay. The scaffold caused formation of an active granulation tissue that shifted to a fibrotic-type connective tissue with accumulation of myofibroblasts and collagen bundles. Most importantly, in transplants with scaffolds, the epithelial-stromal border was normalized developing an ultrastructurally complete basement membrane (BM) including hemidesmosomes. The observed reversion of the tumor phenotype was not due to decreased tumor cell proliferation but correlated with (i) normalization of epidermal differentiation, (ii) condensation of extracellular matrix (ECM) and (iii) reduction of peritumoral protease activity Furthermore, inhibited invasion was paralleled by eliminated tumor vascularization. This was substantiated by a diminished endothelial VEGF-receptor (VEGFR) expression and, in turn, by a concomitant increase in the ECM components thrombospondin-1 (TSP-1) and endostatin, known to impair angiogenesis. Even in transplants of the metastatic high-grade malignant HaCaT-ras A-5RT3 keratinocytes the anti-invasive effect of the scaffold-modulated stroma prevailed. Tumor vascularization and invasion was reduced and the epithelial tissue partially normalized including formation of stretches of BM. This clearly demonstrates that the scaffold-modulated connective tissue not only blocks tumor invasion but reverts the tumor phenotype. These novel findings underline the controlling function of tumor stroma and open new strategies of cancer therapy by targeting tumor stroma elements.

Junctional basement membrane anomalies of skin and mucosa in lipoid proteinosis (hyalinosis cutis et mucosae).

BACKGROUND: Excessive basement membrane (BM) deposition in skin and mucosa is characteristic for lipoid proteinosis (LP; hyalinosis cutis et mucosae), an inherited disease caused by extracellular matrix protein 1 (ECM1) mutations. According to ultrastructure there are striking differences between junctional and microvascular BM. OBJECTIVE: Distinct analysis of the junctional zone in epidermis and oral mucosa, contrasting concentric BM arrays in the microvasculature; evaluation of impact on epithelial histogenesis and differentiation, and specifically on adhesion structures to BM (hemidesmosomes). METHODS: LP-epithelia were analyzed for alterations in differentiation, BM composition and texture, and hemidesmosomal components by indirect immunofluorescence (IIF), electron microscopy (EM), and immunoelectron microscopy (ImEM). RESULTS: Most striking was the irregular deposition of collagen IV and VII, BM-laminin, and laminin-5 at the junctional zone, accompanied by lamellate or punctuated structures below BM (IIF), whereas integrin alpha6beta4 and bullous pemphigoid antigen-1 and -2 (BPAG-1/-2) were regularly aligned. Also integrins alpha2beta1 and alpha3beta1 remained restricted to the epidermal basal layer, while the tissue-specific differentiation markers keratin K1/10 (mucosa, additionally K4/13) appeared delayed indicating mild hyperplasia, further confirmed by focal K6/16 expression. Ultrastructure (EM) disclosed abundance of extended basal cell protrusions and junctional aberrations like exfoliating excessive BM material. Hemidesmosomes were complete, but ImEM indicated weakened interactions between their components (BPAG-1, -2, and HD1). Confirming IIF, collagen IV and VII, and laminin-5 appeared extensively scattered, the latter two probably remaining associated. CONCLUSIONS: Subtle defects in anchorage assembly, spanning the entire BM zone, apparently compromise epithelial-matrix adhesion, which may provoke (mechanical stress-induced) erroneous BM repair.

Lack of nidogen-1 and -2 prevents basement membrane assembly in skin-organotypic coculture.

Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Furthermore, in various locations, extravasation of erythrocytes was observed implying microvascular defects. Mice expressing solely either isoform, had a functional BM, although nidogen-2 binds with lower affinity to the laminin gamma1 chain. Having previously blocked BM formation by interfering with nidogen-1 binding to laminin in skin-organotypic cocultures, here we investigated the roles of nidogen-1 and -2 in this model. For that purpose, human HaCaT cells were grown in three-dimensional cocultures on collagen matrices containing murine fibroblasts of varying nidogen deficiency. As with our experiments blocking laminin-nidogen interaction, lack of both nidogens completely prevented BM deposition and ultrastructural assembly of BM and hemidesmosomes, although other BM proteins remained detectable at comparable levels with no signs of degradation. Supplementation by recombinant nidogen-1 or -2 restored these structures, as shown by immunofluorescence and electron microscopy, confirming that in this system nidogen-2 is equivalent to nidogen-1, and both can promote the development of a functional BM zone.

Epidermal homeostasis in long-term scaffold-enforced skin equivalents.

Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and apoptosis. The lack of appropriate experimental models has largely prevented a better understanding of the regulatory mechanisms controlling epidermal tissue homeostasis in human skin. Keratinocyte culture studies had revealed a strict dependency of regular epidermal differentiation on dermal interactions only accomplishable in three-dimensional skin models. As major drawbacks, conventional models, employing collagen hydrogels as dermal equivalents (DEs) exhibit a rather poor stability and limited lifespan. Here, we present an improved stabilized in vitro-model for long-term growth and differentiation of keratinocytes providing the basis for tissue homeostasis. Keratinocytes were grown on DEs reinforced by modified hyaluronic acid fibers (Hyalograft-3D) and colonized with skin fibroblasts, producing genuine dermis-type matrix. These skin equivalents (SEs) develop superior epidermal architecture with regular differentiation and ultrastructure. Critical aspects of differentiation, still unbalanced in early stages, are renormalized, most strikingly the coexpression of keratins K1/K10, downregulation of regeneration-associated keratins (K16), and restriction of K15 to the basal layer. The strict localization of integrins to basal cells underlining restored tissue polarity, the drop of keratinocyte growth rates towards physiological levels and the rapid formation of a mature basement membrane with abundant anchoring fibrils are altogether features fulfilling the criteria of tissue homeostasis. Therefore, these scaffold-based SEs not only allow for studying homeostasis control but also for the first time provide proper experimental conditions for establishing a stem cell niche in vitro.

Vascular anomalies in lipoid proteinosis (hyalinosis cutis et mucosae): basement membrane components and ultrastructure.

BACKGROUND: In lipoid proteinosis (LP) vascular anomalies represent severe functional defects caused by excessive deposition of basement membrane (BM)-like matrix, particularly around small subepithelial blood vessels. OBJECTIVE: Correlation of microvascular anomalies in morphology and ultrastructure with extracellular matrix composition and cell interactions for elucidating vascular involvement in LP-pathophysiology. METHODS: Biopsies from non-related LP-patients were analyzed by indirect immunofluorescence (IIF), electron microscopy (EM), and immune-EM (ImEM). RESULTS: In LP-skin and mucosa the thickened vessel walls stained strongly for the BM-components type IV collagen, laminin, perlecan, and nidogen (IIF). Integrin alpha6beta4 was regularly collocated with endothelial surface markers such as PECAM (CD31). Ultrastructure (EM) revealed highly ordered matrix deposits around microvessels, with frequently collapsed lumina, functionally compensated by increased vascular density (histology, IIF). Pericytes were trapped between these concentric BM-layers at varying distances towards the periphery (EM), contrasting their regularly close endothelial apposition. Periodic type IV collagen patterns (ImEM) corroborated the multiple BM-leaflet structure and the lack of a common 'fused' endothelial-pericyte BM, seen normally. Presumptive secretory vesicles, abundant in both cell types, implied an equal contribution to BM-synthesis, but also indicated partial loss of endothelial polarity. CONCLUSIONS: In LP thickened vessel walls, composed of multiple BM, profoundly alter microvascular properties, also by interference with endothelial-pericyte interactions. The increased microvascular density reflects compensatory restoration for disabled function. Most remarkable was the exaggerated secretory activity (also at luminal surfaces) underlining the regulatory key role of extracellular matrix protein 1 (ECM1; mutated in LP) in export or turnover of all major BM-components.

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.

Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation complex.

The human repetin gene is a member of the "fused" gene family and localized in the epidermal differentiation complex on chromosome 1q21. The "fused" gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope (CE). Here, we report the isolation and characterization of the human repetin gene and of its protein product. The repetin protein of 784 amino acids contains EF (a structure resembling the E helix-calcium-binding loop-F helix domain of parvalbumin) hands of the S100 type and internal tandem repeats typical for CE precursor proteins, a combination which is characteristic for "fused" proteins. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium, the inner hair root sheat and in the filiform papilli of the tongue. Ultrastructurally, repetin is a component of cytoplasmic non-membrane "keratohyalin" F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin. Finally, we show that EF hands are functional and reversibly bind Ca(2+). Our results indicate that repetin is indeed a member of the fused gene family similar to the prototypical members profilaggrin and trichohyalin.

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.