Justyna Niderla-Bielińska

Potential role of metalloproteinase inhibitors from radiation‑sterilized amnion dressings in the healing of venous leg ulcers

Chronic wounds are a significant socio-economic problem, thus, the improvement of the effectiveness of their treatment is an important objective for public health strategies. The predominant stage of the chronic wound is the inflammatory reaction which is associated with the damage of tissues, possibly due to the excessive secretion and activation of matrix metalloproteinases (MMPs). Several reports have suggested that amnion dressing inhibits tissue destruction and accelerates wound healing. Our recent study revealed that sterilized amnion stimulates keratinocyte proliferation in vitro, while the present study focused on the clinical application of radiation-sterilized amnion in chronic venous leg ulcers and aimed to explain the possible mechanism of its in vivo action. The study involved 25 individuals suffering from venous leg ulceration with a surface area of 10-100 cm2 and a healing rate below 10% per week, as verified during a 2-week screening period. The effectiveness of the amnion dressing was estimated following 4 weeks of treatment. The wound assessment, based on a modified Bates-Jensen Questionnaire, revealed a good and satisfactory response to the treatment in 23 of the 25 patients. The measurement of MMP-2 and MMP-9 activities in wound exudates revealed a decrease in activity in response to amnion application. This effect resulted from the presence of the potent MMP inhibitors, tissue inhibitor of metalloproteinases-1 (TIMP-1), type-1 plasminogen activator inhibitor (PAI-1) and thrombospondin-1 (TSP-1) in the amnion dressings, as shown by real-time fluorescence zymography and protein microarrays. Thus, unlike modern synthetic dressing materials, radiation-sterilized amnion dressings may have a multidirectional beneficial effect on chronic wounds.

Influence of LPS, TNF, TGF-ß1 and IL-4 on the expression of MMPs, TIMPs and selected cytokines in rat synovial membranes incubated in vitro

Synovial membranes are formed by four main types of cells, i.e. fibroblasts, macrophages, epitheliocytes and adipocytes. To study the combined effect of various factors on these cell populations, synovial membranes dissected from rat knee joints were incubated in control medium or medium with lipopolysaccharide (LPS), TNF, TGF-ß1 or IL-4 for 12 h. LPS stimulated TNF secretion and both agents stimulated secretion of IL-6. TGF-ß1 slightly increased IL-6 secretion. LPS increased the mRNA levels of IL-6, IL-1ß, TGF-ß1, MMP1a, MMP1b, MMP3, MMP9, MMP13, MMP14, TIMP1 and TIMP3 while the mRNA levels of MMP2, TIMP2 and TIMP4 were significantly decreased. Expression of IL-1ß, MMP1a, MMP1b, MMP3, MMP9, MMP13 and TIMP1 increased after TNF treatment, while mRNA levels of MMP2, MMP14, TIMP2, TIMP3 and TIMP4 were decreased. TGF-ß1 decreased the mRNA levels of IL-1ß, all MMPs, TIMP1, TIMP2, TIMP4 and increased mRNA levels of itself and TIMP3. IL-4 decreased mRNA levels of IL-1ß, TGF-ß1, MMP2, MMP9, MMP13 and all TIMPs. Only LPS decreased the amount and activity of MMP2. The effect of LPS and cytokines on most of the MMPs and TIMPs produced by whole synovial membrane was in good agreement with previous studies on their action on similar types of cells as those present in synovial membranes, but originating from other tissues. All tested agents decreased MMP2 mRNA expression levels and in the case of LPS also the protein level and its activity determined by zymography, contrary to previous observations on isolated cell populations. This indicates that the response of the organized tissue is an interplay of all components and cannot be deduced from the individual reactions.

Constitutive expression of ligand for natural killer cell NKp44 receptor (NKp44L) by normal human articular chondrocytes

Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.

3-D reconstruction and multiple marker analysis of mouse proepicardial endothelial cell population

BACKGROUND The proepicardium (PE), a transient embryonic structure crucial for the development of the epicardium and heart, contains its own population of endothelial cells (ECs). The aim of our study was to determine the pattern, anatomical orientation and phenotypic marker expression of the endothelial cell network within the PE. RESULTS Immunohistochemical findings revealed that proepicardial ECs express both early and late EC-specific markers such as CD31, Flk-1, Lyve-1 and Tie-2 but not SCL/Tal1, vWF, Dll4 or Notch1. Proepicardial ECs are present in the vicinity of the sinus venosus (SV) and form a continuous network of vascular sprouts/tubules connected with the SV endothelium, with Ter-119-positive erythroblasts in the vascular lumina. CONCLUSIONS On the basis of our results, we postulate the existence of a continuous network of ECs in the PE, exhibiting connection and/or patency with the SV and forming vessels/tubules/strands. Marker expression suggests that ECs are immature and undifferentiated, which was also confirmed with a transmission electron microscopy (TEM) analysis. Our results deliver new data for a better understanding of the nature of proepicardial ECs.

Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes

The goal of this study was to compare expression of chondrocyte-associated antigen (CAA) and cartilage matrix molecules in 2-D (monolayer) and 3-D (Matrigel) culture. Chondrocytes isolated from the cartilage of 3-day-old rats were expanded in monolayer culture for 28 days. CAA expression gradually decreased and was not detected beyond the 96th hour of monolayer culture. Collagen type II and aggrecan mRNA levels decreased during culture. The collagen type I mRNA level increased during the first week and remained high. The increase in the versican mRNA level was less pronounced during the first week and declined slightly after further cultivation. Freshly isolated chondrocytes introduced into Matrigel still expressed CAA after 7 days. Moreover, CAA expression returned in chondrocytes re-cultured in Matrigel after 7 days in monolayer. Similarly, the increase in the mRNA levels of collagen type II and aggrecan in Matrigel was limited to freshly isolated chondrocytes and to those that remained in monolayer for 1 week. Collagen type I mRNA in monolayer and Matrigel cultures of freshly isolated chondrocytes was at a similar level. The introduction of freshly isolated or 7-day monolayer-cultured chondrocytes into Matrigel caused a decrease in the versican mRNA level in comparison with 7- and 14-day 2-D cultures, respectively. On the other hand, chondrocytes seeded in Matrigel after 14 days of monolayer culture did not express CAA, showed decreased levels of collagen type II and aggrecan mRNA and an increase in versican mRNA. In conclusion, it appears that changes in the expression of CAA in 2- and 3-D cultures occur in parallel to changes in typical cartilage matrix molecule expression.

Cardiac mouse lymphatics: developmental and anatomical update.

The adult mouse heart possesses an extensive lymphatic plexus draining predominantly the subepicardium and the outer layer of the myocardial wall. However, the development of this plexus has not been entirely explored, partially because of the lack of suitable methods for its visualization as well as prolonged lymphatic vessel formation that starts prenatally and proceeds during postnatal stages. Also, neither the course nor location of collecting vessels draining lymph from the mouse heart have been precisely characterized. In this article, we report that murine cardiac lymphatic plexus development that is limited prenatally only to the subepicardial area, postnatally proceeds from the subepicardium toward the myocardial wall with the base‐to‐apex gradient; this plexus eventually reaches the outer half of the myocardium with a predominant location around branches of coronary arteries and veins. Based on multiple marker immunostaining, the molecular marker‐phenotype of cardiac lymphatic endothelial cells can be characterized as: Prox‐1+, Lyve‐1+, VEGFR3+, Podoplanin+, VEGFR2+, CD144+, Tie2+, CD31+, vWF−, CD34−, CD133−. There are two major collecting vessels: one draining the right and left ventricles along the left conal vein and running upwards to the left side of the pulmonary trunk and further to the nearest lymph nodes (under the aortic arch and near the trachea), and the other one with its major branch running along the left cardiac vein and further on the surface of the coronary sinus and the left atrium to paratracheal lymph nodes. The extracardiac collectors gain the smooth muscle cell layer during late postnatal stages. Anat Rec, 297:1115–1130, 2014.

Keratinization of outer root sheath cells is prevented by contact with inner root sheath of rat hair follicles

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered—inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca2+ content formed monolayers, which after elevation of Ca2+ produced multilayers with cells containing keratohyalin-like granules. Ca2+ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca2+ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.

Mouse Proepicardium Exhibits a Sprouting Response to Exogenous Proangiogenic Growth Factors in vitro.

Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.

Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

The hair follicle consists of several distinctive epidermal cell layers. The hair root, which undergoes keratinization, is surrounded by two sheaths: the inner root sheath (IRS) and the outer root sheath (ORS). The ORS is continuous with the basal layer of the epidermis. Its cells do not keratinize in situ, unlike IRS. We have previously demonstrated that keratinization of the ORS was prevented by contact with the IRS in hair follicle mid-segments (i.e. fragments dissected from skin at the level above the hair bulb and below the opening of the sebaceous gland duct) cultured on agarose layer. The purpose of this study was to determine whether the same applies to the hair bulb. After isolation, intact bulbs or hair bulb-derived cells were incubated in suspension in a low or high calcium medium. The level of mRNA for differentiation markers: involucrin, filaggrin, keratinocyte differentiation associated protein and trichohyalin, was studied by RealTime PCR. We observed increased Ca(2+) upregulated expression of involucrin, filaggrin, trichohyalin and Kdap in cultures of bulb-derived cells, but in hair bulbs downregulation of involucrin and trichohyalin was observed. We concluded that the inner root sheath exerts an inhibitory effect on the expression of involucrin and trichohyalin already in the hair bulbs. The observation that downregulation of involucrin expression under Ca(2+) influence occurs both in hair bulb and midsegments could simplify future experiments, since their separation does not seem to be necessary.

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by (51)Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.