Mary Ann Stepp

Integrins as receptors for laminins.

Laminins are a family of trimeric glycoproteins present in the extracellular matrix and the major constituents of basement membranes. Integrins are αβ transmembrane receptors that play critical roles in both cell‐matrix and cell‐cell adhesion. Several members of the integrin family, including α1β1, α2β1, α3β1, α6β1 , α7β1 and α6β4 heterodimers serve as laminin receptors on a variety of cell types. This review summarizes recent advances in understanding the involvement of individual integrins in cell interactions with laminins and the roles of laminin‐binding integrins in adhesion‐mediated events in vertebrates, including embryonic development, cell migration and tumor cell invasiveness, cell proliferation and differentiation, as well as basement membrane assembly. We discuss the regulation of integrin function via alternative splicing of cytoplasmic domains of α and β subunits of the integrin receptors for laminins and present examples of functional collaboration between laminin‐binding integrins and non‐integrin laminin receptors. Advances in our understanding of the laminin‐binding integrins continue to demonstrate the essential roles these receptors play in maintaining cell polarity and tissue architecture. Microsc. Res. Tech. 51:280–301, 2000.

Living tissue under treatment of cold plasma atmospheric jet

The interaction of the cold atmospheric plasma jet with fibroblast cells was studied. Plasma jet was initiated in the helium flow blowing through the syringe by application of high ac voltage to the discharge electrodes. The plasma jet had a length of 5cm and a diameter of 1.5–2mm in ambient air. Treatment of cells with plasma jet resulted in decreasing of cell migration rate, cell detachment, and appearance of “frozen” cells, while treatment with helium flow (no plasma) resulted in appearance of frozen cells only. A variety of cellular responses was explained by different intensities of treatment.

Cold atmospheric plasma in cancer therapy

Recent progress in atmospheric plasmas has led to the creation of cold plasmas with ion temperature close to room temperature. This paper outlines recent progress in understanding of cold plasma physics as well as application of cold atmospheric plasma (CAP) in cancer therapy. Varieties of novel plasma diagnostic techniques were developed recently in a quest to understand physics of CAP. It was established that the streamer head charge is about 108 electrons, the electrical field in the head vicinity is about 107 V/m, and the electron density of the streamer column is about 1019 m−3. Both in-vitro and in-vivo studies of CAP action on cancer were performed. It was shown that the cold plasma application selectively eradicates cancer cells in-vitro without damaging normal cells and significantly reduces tumor size in-vivo. Studies indicate that the mechanism of action of cold plasma on cancer cells is related to generation of reactive oxygen species with possible induction of the apoptosis pathway. It is also shown that the cancer cells are more susceptible to the effects of CAP because a greater percentage of cells are in the S phase of the cell cycle.

Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse

Mice lacking syndecan-1 are viable, fertile and have morphologically normal skin, hair and ocular surface epithelia. While studying the response of these mice to corneal epithelial and skin wounding, we identified defects in epithelial cell proliferation and regulation of integrin expression. mRNA profiling of corneal epithelial tissues obtained from wild-type and syndecan-1-/- mice suggest that these defects result from differences in overall gene transcription. In the cornea, syndecan-1-/- epithelial cells migrate more slowly, show reduced localization of α9 integrin during closure of wounds and fail to increase their proliferation rate 24 hours after wounding. In the skin, we did not document a migration defect after full thickness wounds but did observe cell proliferation delays and reduced localization of α9 integrin in the syndecan-1-/- epidermis after dermabrasion. Despite increased cell proliferation rates in the uninjured syndecan-1-/- epidermis and the corneal epithelium, morphologically normal epithelial thickness is maintained prior to injury; however, wounding is accompanied by prolonged hypoplasia in both tissues. Analyses of integrin protein levels in extracts from full thickness skin, revealed increased levels of α3 and α9 integrins both prior to injury and after hair removal in syndecan-1-/- mice but no increase 2 days after dermabrasion. These data for the first time show involvement of α9 integrin in skin wound healing and demonstrate essential roles for syndecan-1 in mediating cell proliferation and regulation of integrin expression in normal and wounded epithelial tissues.

Targeting the cancer cell cycle by cold atmospheric plasma

Cold atmospheric plasma (CAP), a technology based on quasi-neutral ionized gas at low temperatures, is currently being evaluated as a new highly selective alternative addition to existing cancer therapies. Here, we present a first attempt to identify the mechanism of CAP action. CAP induced a robust ~2-fold G2/M increase in two different types of cancer cells with different degrees of tumorigenicity. We hypothesize that the increased sensitivity of cancer cells to CAP treatment is caused by differences in the distribution of cancer cells and normal cells within the cell cycle. The expression of γH2A.X (pSer139), an oxidative stress reporter indicating S-phase damage, is enhanced specifically within CAP treated cells in the S phase of the cell cycle. Together with a significant decrease in EdU-incorporation after CAP, these data suggest that tumorigenic cancer cells are more susceptible to CAP treatment.

In search of markers for the stem cells of the corneal epithelium

The anterior one‐fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.

Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse.

Adult corneal epithelial stem cells (CESCs) have been shown to reside at the periphery of the cornea at a site called the corneoscleral junction or limbus. Although studies have shown that these cells are slow cycling, their molecular characteristics are not well understood. Using a whole‐mount procedure, we show that whereas α9‐integrin is present in a subset of the basal cells at the corneal limbus and absent in the central cornea, β1‐, β4‐, α3‐, and α6‐integrins are more highly expressed overall in central corneal basal cells. To characterize CESCs based on their slow‐cycling nature, we simultaneously evaluated 5‐bromo‐2‐deoxyuridine (BrdU) label‐retaining cells (LRCs) and integrin expression (α9, β1, and β4) in a total of 1,889 cells at the limbus of adult mice that had been injected as neonates with BrdU. Whereas the LRCs were usually observed adjacent to α9‐integrin‐positive cells, most LRCs were α9‐integrin–negative and expressed high levels of β1‐ and β4‐integrin. In addition, we observed more BrdU‐positive LRCs at the superior and inferior quadrants of adult mouse corneas than at the nasal and temporal quadrants, and determined that 0.94 to 3.6% of the limbal basal cells were slow cycling. We conclude from these data that the slow‐cycling LRCs in the adult mouse cornea are enriched in cells that express high levels of β1‐ and β4‐integrin and little α9‐integrin.

Loss of Integrin α9β1 Results in Defects in Proliferation, Causing Poor Re-Epithelialization during Cutaneous Wound Healing

The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and beta1 integrins are important in this process. We previously reported a marked increase in integrin alpha9beta1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte alpha9beta1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin alpha9beta1 knockout mice. We now report that in conditional integrin alpha9 knockout (K14-alpha9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin alpha9beta1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-alpha9 null mice compared with alpha9(fl/-) mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-alpha9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that alpha9beta1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.

The Corneal Epithelial Stem Cell Niche

In recent years, it has become generally accepted that the corneal epithelial stem cells are localized in the basal cell layer of the limbal epithelium. However, a number of questions remain regarding the number, markers, generation, and maintenance of the corneal epithelial stem cells. One of the key questions concerns what makes up the microenvironment or niche that is responsible for allowing the stem cells to remain and function throughout the life of the tissue. This review will consider the unique aspects of the limbus and compare these to what is known about other stem cell niches.

A central role for vimentin in regulating repair function during healing of the lens epithelium

A unique ex vivo mock cataract surgery model is used to study the role of vimentin in repair cell function during wound healing within a clinically relevant setting. Vimentin is found to be critical for the function of repair cells in directing the collective migration of the epithelium during wound healing.