Biomedical subjects

Raija Tammi

Publications and source records attributed to Raija Tammi.

15 recordsLinked to original sources

PubMed2006

A mouse model for Stickler's syndrome: ocular phenotype of mice carrying a targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1).

The influences of targeted heterozygous inactivation of type II (pro)collagen gene (Col2a1) on eye structures in the 15-month-old C57BL/6JOlaHsd mouse was studied. The eyes were collected from C57BL mice heterozygous for a targeted inactivation of one allele of the Col2a1 gene (Col2a1(+/-) mice). The eyes of C57BL mice with normal gene alleles were used as controls (Col2a1(+/+) mice). Ocular histology was analyzed from tissue sections, stained with hematoxylin and eosin, toluidine blue and alcian blue. Type II collagen was localized by immunohistochemistry. Hyaluronan (HA) was stained utilizing the biotinylated complex of the hyaluronan-binding region of aggrecan and link protein (bHABC). The anterior segment of the eye was well-formed in both genotypes, but typical folding of ciliary processes was decreased, while increased stromal extracellular matrix vacuolization was seen in the Col2a1(+/-) mice. In the lens of these mice, subcapsular extracellular matrix changes were observed. Differences in retinal structures or the number of the eyes with retinal detachment were not detected between the genotypes. In Col2a1(+/-) mice, staining for type II collagen was weaker in cornea, ciliary body, iris, lens, vitreous, retina, choroid and sclera than in the control mice. HA staining was detected in the extraocular tissues, ciliary body, iris and the choroid of both genotypes. HA staining was observed only in the vitreous body of the control animals. Heterozygous inactivation of Col2a1 gene causes structural defects in the murine eye. The observed structural changes in the ciliary body, lens and vitreous of the Col2a1(+/-) mice may represent ocular features found in the human Stickler syndrome, where the abnormalities result from COL2A1 gene mutations which lead to functional haploinsufficiency.

Aggrecans↗

PubMed2005

Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing.

The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1alpha release) were verified at both time points and correlated to PII values. The best correlation (R(2) = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1alpha release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicals were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.

Animals↗

PubMed2005

The human hyaluronan synthase 2 gene is a primary retinoic acid and epidermal growth factor responding gene.

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis and subject to large concentration changes associated with keratinocyte proliferation, migration, and differentiation induced by paracrine and endocrine factors like epidermal growth factor (EGF) and all-trans-retinoic acid (RA). We found that in REK cells EGF and all-trans-RA up-regulated hyaluronan synthase 2 (Has2) gene expression within 2 h 4-fold each and in HaCaT human immortal keratinocytes 8- and 33-fold, respectively. The first 10 kb of the human Has2 promoter were scanned in silico and in vitro for potential response elements of signal transducer and activator of transcription (STAT) or RA receptor (RAR) proteins. We identified a STAT-response element in the proximal promoter region and confirmed its functionality in response to EGF by chromatin immunoprecipitation (ChIP) assays. Direct in vitro binding of RARs to four RARE candidates within the Has2 promoter could not be observed at stringent gel shift conditions, but reporter gene assays demonstrated functionality of a complex of two of these RAREs located approximately 1200 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RA-dependent binding of RAR, retinoid X receptor, mediator protein, and RNA polymerase II and also histone 4 acetylation to a promoter region containing the complex RARE. Taken together, the human Has2 gene is a potent primary EGF and all-trans-RA responding gene with a complex regulation.

Acetylation↗

PubMed2005

Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury.

Hyaluronan, a major extracellular matrix component in the epidermis, has been shown to control keratinocyte proliferation and differentiation in vitro. We assayed hyaluronan and hyaluronan synthases (has1-3) in mouse epidermis during fetal development, postnatal life, and trauma reaction in vivo. Hyaluronan increased in the epidermis when keratinocytes started to stratify on day E15, remained high until birth, and then rapidly declined, with corresponding changes in the mRNA levels of has2 and has3. The hyaluronan in adult mouse epidermis mainly resided around the orifices of the hair follicles, and the overall concentration was about one order of magnitude lower than in adult human epidermis. In adult mice, epidermal trauma caused by tape stripping rapidly increased hyaluronan, leading to a 6-fold increase in epidermal hyaluronan on day 3 following trauma. The hyaluronan response was associated with a strong induction of has2 and has3 mRNA, slightly higher CD44 expression, and considerable epidermal hyperplasia. The data show that the pre- and postnatal fluctuations in epidermal hyaluronan content correlate with the expression levels of has2 and has3. Stimulated hyaluronan synthesis through upregulated has expression is an inherent feature of the keratinocyte activation triggered by tissue trauma, and presumably important for a proper healing response.

Age Factors↗

PubMed2004

Expression of extracellular matrix components versican, chondroitin sulfate, tenascin, and hyaluronan, and their association with disease outcome in node-negative breast cancer.

PURPOSE: The purpose is to determine whether the levels of expression of extracellular matrix components in peritumoral stroma are predictive of disease outcome for women with node-negative breast cancer. EXPERIMENTAL DESIGN: Tumor tissue from 86 patients with node-negative breast cancer was examined by immunohistochemical staining for the expression of versican, chondroitin sulfate (CS), tenascin, and hyaluronan (HA). With the exception of HA, the expression of the extracellular matrix components was measured by video image analysis. Statistical correlation of the immunohistochemical data with clinicopathological characteristics and disease outcome was performed. RESULTS: All of the extracellular matrix components were present in the peritumoral stroma of the entire study cohort. In contrast, immunoreactivity within the cancer cell was observed in 82% of tumors for HA, 12% for CS, and 4% for tenascin; no immunostaining of cancer cells for versican was observed for any of the tumors. Cox regression and Kaplan-Meier analyses indicated that elevated expression of stromal versican predicted increased risk and rate of relapse in this cohort. Elevated expression of tenascin was predictive of increased risk and rate of death only. Although neither CS nor HA were predictive of disease outcome in this cohort, tumor size was predictive of increased risk and rate of both relapse and survival. CONCLUSIONS: Elevated expression within peritumoral stromal matrix of versican and tenascin was predictive of relapse-free and overall survival, respectively, in women with node-negative breast cancer.

Adult↗

PubMed2004

Genetic alterations in the peritumoral stromal cells of malignant and borderline epithelial ovarian tumors as indicated by allelic imbalance on chromosome 3p.

Stromal accumulation of hyaluronan in epithelial ovarian cancers is an independent predictor of tumor spreading and unfavorable outcome of the disease. We started to screen for chromosomal causes of this accumulation by studying deletions in 3p21.3, a region harboring 3 hyaluronidase genes (HYAL1-3) among other potentially important tumor suppressors. Using 6 microsatellite markers from this region, allelic imbalance was found in 60-87% of the informative tumor cells microdissected from histologic sections of 58 patients with epithelial ovarian cancer. However, adjacent stromal cells originally intended as controls showed allelic imbalance at a frequency almost as high as the tumor cells (52-80%). A further laser capture microdissection on 10 borderline tumors also showed a high rate of allelic imbalance, both in the epithelial and stromal cells, but with a pattern slightly different from cancers. Allelic imbalance in the tumor epithelium or stroma was not correlated with the accumulation of hyaluronan or clinicopathologic parameters, including tumor stage and grade. The results suggest that factors other than inactivation of the HYAL1-3 genes are responsible for hyaluronan accumulation in epithelial ovarian tumors. Moreover, the results indicate that the stromal cells of the epithelial ovarian cancers not only respond to the signals from malignant epithelium but also have themselves undergone genetic alterations in markers partly identical to those in the cancer epithelial cells and may actively contribute to the development of the tumor from its early stages to the late determinants of patient mortality.

Allelic Imbalance↗

PubMed2004

The hyaluronan synthesis inhibitor 4-methylumbelliferone prevents keratinocyte activation and epidermal hyperproliferation induced by epidermal growth factor.

Since excessive epidermal hyaluronan is associated with hyperproliferative states and disturbed terminal differentiation of the keratinocytes, we hypothesized that 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, could counteract these phenotypic features. Cultured epidermal keratinocytes showed a concentration dependent, maximum 83% reduction of hyaluronan in the presence of 0.2-1.0 mM 4-MU, whereas less decline was seen in the synthesis of chondroitin and heparan sulfate. The reduced hyaluronan was associated with no apparent change in its molecular mass. The 4-MU-treated keratinocytes showed an accentuated epithelial morphology with a flat, round cell shape, increased stress fibers and large vinculin-positive adhesion plaques, cytoskeletal changes consistent with the markedly reduced migration rate observed in scratched monolayer cultures. High concentrations of 4-MU also caused a block in keratinocyte proliferation, reversible upon 4-MU withdrawal. In the epidermis of organotypic cultures, 4-MU prevented the hyaluronan accumulation and epidermal hypertrophy induced by epidermal growth factor. The present results concur with earlier data indicating that enhanced cell locomotion and proliferation are associated with hyaluronan synthesis in activated keratinocytes. Cell proliferation, however, was blocked more strongly than expected on the basis of the incomplete hyaluronan synthesis inhibition, and may represent a novel target of 4-MU. At any rate, 4-MU and equivalent hyaluronan synthesis inhibitors might be considered for situations where suppression of epidermal activation and hyperproliferation is warranted.

Animals↗

PubMed2003

Versican in epithelial ovarian cancer: relation to hyaluronan, clinicopathologic factors and prognosis.

Versican, a proteoglycan previously reported to increase in other malignant tumours, was studied immunohistochemically in 299 primary epithelial ovarian cancers, their 43 metastases and 6 normal ovaries to evaluate its prognostic value and relation to hyaluronan, another extracellular matrix molecule increased in cancer and a binding partner of versican. The stainings were scored according to the area percentage of strong versican signal of total peri- and intratumoural stroma as low (<15%) or high (>or=15%). Epithelial staining of the tumours was scored as positive or negative. Low and high area percentage of strong stromal versican staining were observed in 133 and 166 carcinomas, respectively. A low area percentage of strong stromal versican staining correlated with mucinous histology (p = 0.019) and early International Federation of Gynecologists and Obstetritians (FIGO) stage (p < 0.0005), whereas a high percentage was associated with reduced 5-year survival rate of the patients (44% vs. 32%; p = 0.032). Versican was associated with the cancer cells in 151 tumours and correlated with clear cell histology (p < 0.0005), early FIGO stage (p = 0.049) and increased recurrence-free survival (63% vs. 47%; p = 0.032). However, in Cox's multivariate analyses with the conventional prognostic factors included, neither stromal nor cancer cell-associated versican reached a significant prognostic value. Versican is thus enriched in the malignant stroma surrounding and promoting the growth of ovarian cancer, probably acting with hyaluronan, and associates with unfavourable prognosis but does not constitute an independent indicator of patient survival.

Adolescent↗

PubMed2003

Parathyroid hormone rapidly stimulates hyaluronan synthesis by periosteal osteoblasts in the tibial diaphysis of the growing rat.

Short term treatment (3-24 h) with parathyroid hormone (PTH) stimulated the synthesis and accumulation of hyaluronan (HyA) in explant cultures of tibial diaphyses from young rats. PTH increased the overall HyA content of periosteum 5-fold, with the basal cambium layer exhibiting the greatest enhancement ( approximately 8-fold). PTH increased the HyA content of cortical bone by 2-fold while not affecting the HyA content of bone marrow. PTH treatment greatly enhanced HyA staining throughout all layers of the periosteum, although its most dramatic effect occurred in the basal cambium layer. Here, unlike in the control tissue sections, nearly all cambium-lining osteoblasts stained intensely positive for HyA. PTH treatment enhanced the HyA staining of osteocytes in cortical bone tissue sections to the extent that the lacunocanalicular system became visualized. Three significant findings were revealed in this study. First, mature periosteal osteoblasts, under natural conditions, do not contain much HyA in their surrounding extracellular matrix but dramatically enhance their matrix HyA content when treated with PTH. Second, pre-osteocytes and osteocytes contain more HyA in their natural matrix than mature lining osteoblasts, and they appear to have functional PTH receptors because they responded to PTH treatment with an enhancement of HyA content. Finally, it was observed that the lining cells along the endosteal surface of the diaphysis did not stain strongly positive for HyA either naturally or when exposed to PTH treatment. This indicates that periosteal and endosteal osteoblastic cell populations exhibit metabolic differences in their extracellular matrix responses to PTH.

Animals↗

PubMed2003

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.

Animals↗

PubMed2003

Epidermal cell culture model derived from rat keratinocytes with permeability characteristics comparable to human cadaver skin.

The permeability characteristics of an organotypic epidermal culture model derived from rat epidermal keratinocytes, ROC, and isolated human cadaver epidermis, HEM, were compared. Rat epidermal keratinocyte (REK) cell line was grown for 3 weeks on collagen gel in the absence of feeder cells in culture inserts at an air-liquid interface. Transdermal permeabilities of 18 compounds ranging from 92 to 504 in molecular weight and from -4.3 to 3.9 in log of octanol-water partition coefficient, charged or uncharged, were measured in the culture model and isolated human epidermis. The REK organotypic culture model (ROC) provided a close estimate of human epidermal permeabilities over the whole range of the solutes used with on the average of 2-fold higher permeability coefficients (range 0.3-5.2) than those obtained from isolated human cadaver epidermis. The easily maintained and reproducible ROC model may be useful in screening transepidermal drug permeabilities together with possessing potential for research on dermal formulations, irritation, toxicity and gene therapy.

Animals↗

PubMed2003

EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.

Animals↗

PubMed2002

Elevated hyaluronan concentration without hyaluronidase activation in malignant epithelial ovarian tumors.

The concentration and histological distribution of hyaluronan, a tumor promoting extracellular matrix polysaccharide, and the activity of hyaluronidase, a potential source of angiogenic hyaluronan oligosaccharides, were analyzed in malignant epithelial (n = 24), borderline (n = 8), benign epithelial (n = 20), functional cyst (n = 21), and normal (n = 5) tissue samples of human ovary. Hyaluronan concentration increased specifically in cancers (P = 0.001), particularly in grade 3 tumors (>49-fold) and in metastases (>89-fold). Hyaluronan staining in the tissues correlated with hyaluronan concentration (P = 0.002). Hyaluronidase activity slightly decreased from semimalignant through low grade to high grade tumors (P = 0.041). Therefore, hyaluronan accumulation, but not hyaluronidase activation, is associated with the aggressiveness of ovarian epithelial cancer.

Adult↗

PubMed2002

Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.

Hyaluronan is a major component of the epidermal extracellular matrix, is actively synthesized by keratinocytes and shows fast matrix turnover in the stratified epithelium. We probed the importance of hyaluronan synthesis in keratinocytes by establishing cell lines carrying the exogenous hyaluronan synthase 2 (Has2) gene in sense and antisense orientations to increase and decrease their hyaluronan synthesis, respectively. Compared with cell lines transfected with the vector only, most clones containing the Has2 sense gene migrated faster in an in vitro wounding assay, whereas Has2 antisense cells migrated more slowly. Has2 antisense clones showed delayed entry into the S phase of cell cycle following plating, smaller lamellipodia and less spreading on the substratum. The decrease of hyaluronan on the undersurface of Has2 antisense cells was associated with an increased area of adhesion plaques containing vinculin. Exogenous hyaluronan added to the keratinocyte cultures had a minor stimulatory effect on migration after wounding but did not restore the reduced migratory ability of Has2 antisense cells. Hyaluronan decasaccharides that displace receptor bound hyaluronan in keratinocytes, and Streptomyces hyaluronidase sufficient to remove most cell surface hyaluronan had little effect on cell migration. The results suggest that the dynamic synthesis of hyaluronan directed by Has2, rather than the abundance of pericellular hyaluronan, controls keratinocyte migration, a cell function vital for the repair of squamous epithelia following wounding.

Animals↗

PubMed2002

Hyaluronan expression in differentiated thyroid carcinoma.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation, and proliferation, and is supposed to contribute to the spreading of several human cancers. Little is known about the role of HA in the development and progression of differentiated thyroid carcinoma (DTC). The expression and prognostic value of HA were therefore evaluated in 204 consecutive patients with DTC. A biotinylated affinity probe specific for HA was applied to paraffin-embedded tumour samples to assay the expression of HA in carcinoma cells and in intra/peritumoural stroma. In a majority of the samples, a high percentage (>or=90%) of normal thyroid follicle epithelial cells were HA-positive. This high percentage was also found in 80 (47%) papillary carcinomas, but only in seven (21%) follicular carcinomas (p=0.004). Age (>60 years) of the patients was significantly associated with a low percentage of HA-positive cancer cells (p=0.013). Cancer cell-associated HA correlated significantly with the percentage of cells expressing total CD44 and its isoforms containing exons v3 and v6 (r=0.223-0.289, p<0.001 for all). The tumour stroma was always positive for HA. Stromal staining intensity did not differ markedly between papillary and follicular carcinomas. A strong stromal HA staining intensity was related to distant metastases (p=0.044), high pTNM stage (p=0.024), old age (>60 years) (p=0.043), and cancer-related mortality (p=0.001). In a log-rank univariate survival analysis, strong stromal HA staining intensity was related to DTC mortality (p=0.0007). Cancer cell-associated HA expression did not significantly correlate with patient survival. In Cox's multivariate survival analysis, age (>60 years, p=0.0164), gender (p=0.0251), and pTNM stage (p=0.0121) were significant independent prognostic factors for DTC-related death. These results suggest that strong stromal HA staining intensity is related to progression and unfavourable outcome in DTC patients, while the clinical factors remain more powerful in predicting DTC-related death.

Adolescent↗