Ernst Reichmann

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion

As a novel approach to studying the modulation of the polarized epithelial phenotype, we have expressed c-Fos and c-Myc estrogen receptor fusion proteins (c-FosER and c-MycER) in mammary epithelial cells. The hybrid proteins could be activated by estrogen for defined time periods and after the cells had achieved their fully polarized organization. Activation of c-MycER deregulated proliferation but did not affect epithelial polarity. Short-term activation of c-FosER induced the reversible loss of morphological and functional cell polarity. In contrast, long-term stimulation of c-FosER caused the cells to depolarize irreversibly, to invade collagen gels, and to undergo epithelial-fibroblastoid cell conversion. Our data suggest that Fos proteins are important in modulating the epithelial phenotype both in normal tissue development and in invasive processes.

Tissue engineering of skin

The engineering of skin substitutes and their application on human patients has become a reality. However, cell biologists, biochemists, technical engineers, and surgeons are still struggling with the generation of complex skin substitutes that can readily be transplanted in large quantities, possibly in only one surgical intervention and without significant scarring. Constructing a dermo-epidermal substitute that rapidly vascularizes, optimally supports a stratifying epidermal graft on a biodegradable matrix, and that can be conveniently handled by the surgeon, is now the ambitious goal. After all, this goal has to be reached coping with strict safety requirements and the harsh rules of the economic market.

Tumor cell invasiveness correlates with changes in integrin expression and localization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-β (TGFβ1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits β1, α2 and α3, or α5 and α6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits β1 and α6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and α5 at the entire plasma membrane. During EMT, EpRas cells formed an α5β1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking α5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFβ1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional α5β1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the α6β4 ligand laminin 5, suggesting that α6β4 is no longer functional after EMT and replaced by mesenchymal integrins such as α5β1.

Markers to Evaluate the Quality and Self-Renewing Potential of Engineered Human Skin Substitutes In Vitro and after Transplantation

We screened a series of antibodies for their exclusive binding to the human hair follicle bulge. In a second step these antibodies were to be used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. Of all the antibodies screened, we identified only one, designated C8/144B, that exclusively recognized the hair follicle bulge. However, C8/144B-binding cells were never detected in the human epidermal stratum basale. In the bulge C8/144B-binding cells gave rise to cytokeratin 19-positive cells, which were also tracked in the outer root sheath between bulge and the hair follicle matrix. Remarkably, cytokeratin 19-expressing cells were never detected in the hair follicle infundibulum. Yet, cytokeratin 19-expressing keratinocytes were found in the epidermal stratum basale of normal skin as a subpopulation of cytokeratin 15-positive (not C8/144B-positive) basal keratinocytes. Cytokeratin 19/cytokeratin 15-positive keratinocytes decreased significantly with age. We suggest that cytokeratin 19-expressing cells represent a subpopulation of basal keratinocytes in neonates and young children (up to 1.5 years) that is particularly adapted to the lateral expansion of growing skin. Our data show that cytokeratin 19 in combination with cytokeratin 15 is an important marker to routinely monitor epidermal homeostasis and (at least indirectly) the self-renewing potential of engineered skin.

The biological role of the Fas/FasL system during tumor formation and progression.

The cells of an organism are constantly exposed to conflicting environmental cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. This default death pathway appears to be activated by dedicated death receptors such as Fas, the TRAIL-receptors and other tumor necrosis factor receptor superfamily proteins (TNFR SFPs). Our understanding of how these counteracting receptor systems are modulated during tumorigenesis is only moderate. Nevertheless, there is now broad evidence that susceptibility of tumor cells towards Fas-mediated apoptosis is largely reduced. In addition, tumor cells frequently exhibit de novo expression of Fas-ligand (FasL) which plays a significant role in local tissue destruction, metastatic spread and immune escape of the tumor cells. Restoring the apoptotic potential of cancer cells upon modulating the expression and activity of certain key components of the cell death machinery is an attractive and obvious therapeutic anti-cancer strategy.

Matriderm versus Integra: a comparative experimental study.

AIM To compare engraftment rates and vascularisation in a rat model using either Integra Artificial Skin or Matriderm. METHODS Matriderm and the dermal part of Integra were compared in a two-step procedure including matrix implantation and subsequent epidermal grafting. Neonatal rat epidermis was used as coverage to test for rapid and complete take. RESULTS Efficiency and quality of vascularisation expressed by take rate of epidermis, and thickness of resulting neodermis, were identical for both matrices. CONCLUSION This first comparison of Matriderm with Integra in a rat model revealed no major differences in engraftment rates or vascularisation.

Transforming growth factor‐β and Ras regulate the VEGF/VEGF‐receptor system during tumor angiogenesis

The formation of new microvasculature by capillary sprouting, or angiogenesis, is a prerequisite for solid tumor growth. The genetic alterations required to activate the angiogenic program in tumor angiogenesis are still only vaguely known, but dominantly acting oncoproteins may have a much greater impact than previously realized. Here we have studied the consequences of oncogenic transformation on tumor angiogenesis in a mouse mammary carcinoma model. We provide evidence that the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptor‐2 (Flk‐1), a signaling system centrally involved in tumor angiogenesis, occurs efficiently in tumors formed by Ras‐transformed mammary epithelial cells and that both TGF‐β1 and hypoxia are potent inducers of VEGF expression in these cells. VEGF induction in the tumor periphery is mainly triggered by TGF‐β1, whereas VEGF expression in perinecrotic areas is regulated by both hypoxia and TGF‐β1. As the Ras‐transformed tumor cells convert into migrating, fibroblastoid cells that start to produce TGF‐β during tumor progression, the TGF‐β effect on VEGF expression becomes propagated throughout the tumor tissue. Thus, in progressed tumors, areas of TGF‐β1 activation and hypoxia may overlap and hence cooperate to induce VEGF expression and angiogenesis. Nevertheless, the overexpression of VEGF in non‐Ras‐transformed mouse mammary epithelial cells was not sufficient to promote vascularization in vivo. Based on these findings, we conclude that amongst the multiple mutations that render a normal cell tumorigenic, oncogenic Ras is a major player that in conjunction with the tumors micro‐environment sets the stage for tumor cell invasion and angiogenesis.

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells.

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipients vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.

Glucose sensing in human epidermis using mid-infrared photoacoustic detection

No reliable non-invasive glucose monitoring devices are currently available. We implemented a mid-infrared (MIR) photoacoustic (PA) setup to track glucose in vitro in deep epidermal layers, which represents a significant step towards non-invasive in vivo glucose measurements using MIR light. An external-cavity quantum-cascade laser (1010–1095 cm−1) and a PA cell of only 78 mm3 volume were employed to monitor glucose in epidermal skin. Skin samples are characterized by a high water content. Such samples investigated with an open-ended PA cell lead to varying conditions in the PA chamber (i.e., change of light absorption or relative humidity) and cause unstable signals. To circumvent variations in relative humidity and possible water condensation, the PA chamber was constantly ventilated by a 10 sccm N2 flow. By bringing the epidermal skin samples in contact with aqueous glucose solutions with different concentrations (i.e., 0.1–10 g/dl), the glucose concentration in the skin sample was varied through passive diffusion. The achieved detection limit for glucose in epidermal skin is 100 mg/dl (SNR=1). Although this lies within the human physiological range (30–500 mg/dl) further improvements are necessary to non-invasively monitor glucose levels of diabetes patients. Furthermore spectra of epidermal tissue with and without glucose content have been recorded with the tunable quantum-cascade laser, indicating that epidermal constituents do not impair glucose detection.

Bioengineering Dermo-Epidermal Skin Grafts with Blood and Lymphatic Capillaries

Human lymphatic capillaries were engineered in a 3D hydrogel system to improve dermo-epidermal skin grafting. Skin Grafts Need Plumbing, Too To help heal a severe burn or wound, clinicians surgically transplant skin grafts, which consist of the epidermis (outer skin layer) and, often, part of the dermis (deeper layer, directly below the epidermis). The success of these grafts, however, is limited by the ability of blood vessels to form in the newly transplanted skin and deliver nutrients to the cells. Research has also suggested that the lymphatics may be necessary for skin graft survival, by essentially draining immune cells, debris, and excess fluid from the wounded area. Here, Marino, Luginbühl, and colleagues engineered a skin graft that wasn’t just the patient’s skin cells—it also contained both lymph and blood capillaries “prevascularized” ex vivo and then transplanted onto a wound. The authors created the dermo-epidermal skin grafts by taking cells from human foreskin, called human dermal microvascular endothelial cells (HDMECs), and embedding them in three-dimensional hydrogels. HDMECs consist of a mixture of both lymphatic endothelial cells and blood vessel endothelial cells, so both types of functional capillaries—blood and lymph—formed from these cells in vitro in the fibrin or collagen hydrogels. Moving in vivo, the authors transplanted engineered skin grafts containing the HDMECs as well as human fibroblasts and keratinocyes—two cell types found in skin—onto the wounded backs of nude rats (animals without an immune system). Marino, Luginbühl, et al. reported that the human skin grafts formed the expected skin layers after 2 weeks, connected with existing rat lymphatic capillaries, and drained fluid away from the wound. Although testing and characterization are still needed in animals with an immune system and with skin similar to humans (such as a pig), these engineered dermo-epidermal hydrogels potentially represent the next generation of skin grafts, complete with the vascular and lymphatic plumbing and ready to transplant. The first bioengineered, autologous, dermo-epidermal skin grafts are presently undergoing clinical trials; hence, it is reasonable to envisage the next clinical step at the forefront of plastic and burn surgery, which is the generation of autologous skin grafts that contain vascular plexuses, preformed in vitro. As the importance of the blood, and particularly the lymphatic vascular system, is increasingly recognized, it is attractive to engineer both human blood and lymphatic vessels in one tissue or organ graft. We show here that functional lymphatic capillaries can be generated using three-dimensional hydrogels. Like normal lymphatics, these capillaries branch, form lumen, and take up fluid in vitro and in vivo after transplantation onto immunocompromised rodents. Formation of lymphatic capillaries could be modulated by both lymphangiogenic and anti-lymphangiogenic stimuli, demonstrating the potential usefulness of this system for in vitro testing. Blood and lymphatic endothelial cells never intermixed during vessel development, nor did blood and lymphatic capillaries anastomose under the described circumstances. After transplantation of the engineered grafts, the human lymphatic capillaries anastomosed to the nude rat’s lymphatic plexus and supported fluid drainage. Successful preclinical results suggest that these skin grafts could be applied on patients suffering from severe skin defects.