Specific TRPC6 channel activation, a novel approach to stimulate keratinocyte differentiation.

The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca(2+) influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca(2+) concentration ([Ca(2+)](o)), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca(2+) influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca(2+)- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca(2+)](o). Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation.

Diminished thrombus formation and alleviation of myocardial infarction and reperfusion injury through antibody- or small-molecule-mediated inhibition of selectin-dependent platelet functions.

BACKGROUND AND OBJECTIVES: P-selectin ctin has been implicated in important platelet functions. However, neither its role in thrombus formation and cardiovascular disorders nor its suitability as a therapeutic target structure is entirely clear. DESIGN AND METHODS: Platelet aggregation was assessed in complementary in vitro settings by measurements of static aggregation, standardized aggregometry and dynamic flow chamber assays. Degradation of aggregates was also analyzed under flow conditions using video microscopy. In vivo, platelet rolling in cutaneous venules was assessed by intravital microscopy in wild-type mice treated with selectin-blocking compounds as well as in P-selectin-deficient mice. FeCl3-induced arterial thrombosis was studied by intravital microscopy in untreated mice or mice treated with an inhibitor of selectin functions. Finally, inhibition of selectin functions was studied in an ischemia/reperfusion injury model in rats. RESULTS: Antibody- or small-molecule-mediated inhibition of P-selectin functions significantly diminished platelet aggregation (p<0.03) and platelet-neutrophil adhesion in vitro (p<0.01) as well as platelet aggregate sizes under flow (p<0.03). Established aggregates were degraded, either via detachment of single platelets following addition of efomycine M, or via detachment of multicellular clumps when P-selectin-directed Fab-fragments were used. In vivo, selectin inhibition resulted in a greater than 50% reduction of platelet rolling in cutaneous venules (p<0.01), producing rolling fractions similar to those observed in P-selectin-deficient mice (p<0.05). Moreover, inhibition of selectin functions significantly decreased the thrombus size in FeCl3-induced arterial thrombosis in mice (p<0.05). In an ischemia/reperfusion injury model in rats, small-molecule-mediated selectin inhibition significantly reduced myocardial infarct size from 18.9% to 9.42% (p<0.001) and reperfusion injury (p<0.001). INTERPRETATION AND CONCLUSIONS: Inhibition of P-selectin functions reduces platelet aggregation and can alleviate platelet-related disorders in disease-relevant preclinical settings.

Diminished lymphocyte adhesion and alleviation of allergic responses by small-molecule- or antibody-mediated inhibition of L-selectin functions.

Selectins are attractive targets for specific anti-inflammatory therapies. Using human lymphocytes as well as an L-selectin-transfected pre-B-cell line in dynamic flow chamber experiments, we could demonstrate that the small-molecule compound efomycine M blocks L-selectin-mediated lymphocyte rolling on sialylated Lewis(X), an action that was confirmed by plasmon resonance spectroscopy. Recruitment of naive lymphocytes to peripheral lymph nodes depends on L-selectin-mediated adhesion to high endothelial venules. We performed intravital microscopy studying lymphocyte rolling in peripheral lymph nodes and showed a 53% reduction (P=0.0006) of lymphocyte rolling in mice treated with efomycine M or a function-blocking antibody against L-selectin. In addition, the number of lymph node-homing T cells was reduced by >60% using either efomycine M or L-selectin-blocking antibodies. As recruitment of naive lymphocytes is a prerequisite for sensitization in T-cell-mediated immune reactions and allergic responses, mice were treated with efomycine M or an L-selectin-specific antibody during contact sensitization with DNFB. After adoptive transfer of corresponding T cells into non-sensitized recipient mice, the capacity of these cells to induce contact hypersensitivity was significantly reduced (P=0.0002 and P=0.0001, respectively). Our data demonstrate that it is possible, in principle, to diminish T-cell-mediated allergic reactions through interference with L-selectin functions during the early sensitization phase.

Structure-function relation of efomycines, a family of small-molecule inhibitors of selectin functions.

Selectin-mediated leukocyte adhesion to endothelia, the crucial first step initiating the pathogenic cascade of inflammation, is an attractive target for specific therapies. The small-molecule macrolide, efomycine M, inhibits selectin-mediated leukocyte adhesion in vitro and in vivo, and effectively alleviates inflammatory disorders in vivo. To define the molecular basis of the therapeutically relevant antiadhesive properties of efomycines, several new species of this family were purified and/or synthesized. Efomycines E and G were isolated from Steptomyces BS1261. Efomycine O was synthesized by Lewis acid-catalyzed acetalization and efomycine M was generated by base-catalyzed deglycosylation. Efomycine S resulted from ester cleavage of the macrolide ring system, and efomycine T represents the peracetylated form of efomycine M. When the functional activity of efomycines on adhesion of leukocytes to vascular endothelium was studied, some remarkable differences between the compounds became apparent, inasmuch as efomycines E, G, M, and O significantly inhibited adhesion of both human and porcine leukocytes to the vascular endothelium, whereas efomycines S and T did not show any biological activity. A novel docking engine (ProPose), generating an improved, fully configurable protein-ligand interaction model, demonstrated that biological activities of efomycines can be predicted in silico, thus highlighting the utility of such combinatorial approaches.

Psoriasis: Clinical manifestations, pathogenesis and therapeutic perspectives.

Extract: Psoriasis, a common inflammatory skin disorder, has recently moved into the limelight of both basic and clinical research. On the one hand, research into its pathogenesis has furthered our general understanding of T cell-mediated autoimmune disorders, and, on the other hand, psoriasis is used increasingly as a primary target disorder for novel therapies that are pathogenesis-oriented. Given that psoriasis affects approximately 2% of the population, it is a truly common skin disease. It is, therefore, somewhat surprising that the first description of psoriasis as a distinct entity dates back only to the year 1841. Geographic and ethnic factors appear to have a significant influence on the prevalence of psoriasis: ranges from 0% in the population of the Pacific islands of Samoa to 12% in the Arctic Kasach'ye have been reported. Ethnic influence is particularly evident when looking at the prevalence in African Americans, which is less than half that of the United States in general. Numerous family studies have provided compelling evidence for a genetic predisposition to develop psoriasis, although the inheritance pattern is still unclear. Genome-wide linkage studies have identified several putative psoriasis susceptibility loci, one of which located in the MHC (major histocompatibility complex, a cluster/locus of genes involved in the immune response of rejection) region on chromosome 6 was found to be present in several populations. This locus, termed "PSORiasis Susceptibility 1" (PSORS1), can thus be considered the major susceptibility locus and is associated with up to 50% of psoriasis cases.

Targeting selectin functions in the therapy of psoriasis.

As leukocytes play a primary role in the immunopathogenesis of psoriasis, it is a reasonable assumption that preventing those cells from localizing to the sites of cutaneous inflammation can stop the disease process. Selectins, a family of three single-chain transmembrane adhesion molecules, which bind to carbohydrate moieties displayed on other cells, are pivotally involved in the initial steps of leukocyte recruitment, i.e. tethering and rolling along the endothelial lining of blood vessels. Thus, compounds impairing selectin functions have been developed to treat inflammatory disorders, such as psoriasis. Potential strategies to interfere with selectin functions include direct inhibition through monoclonal antibodies or small-molecule compounds, transcriptional regulation of selectin expression, and modulation of the post-transcriptional glycosylation of selectin ligands. More than a dozen different compounds targeting selectin functions are currently under development, several of which have shown promising effects on leukocyte recruitment and the therapy of inflammatory conditions under experimental conditions. However, based on preclinical and early clinical investigations, it appears that in some cases specifically targeting the function of single selectins may not be sufficient to effectively interrupt the inflammatory cascade. This is, at least in part, due to considerable redundancies and overlaps in the functions of the selectins, a notion that is corroborated by the apparently more pronounced therapeutic efficacy of some compounds with broader activity against several selectins. Overall, while targeting selectin functions promises rather selective and pathogenesis-based therapeutic approaches against psoriasis, the clinical value of such strategies, alone or in combination with other therapies, remains to be seen.

The molecular basis of lymphocyte recruitment to the skin: clues for pathogenesis and selective therapies of inflammatory disorders.

Spatial compartmentalization and tissue-selective localization of T lymphocytes to the skin are crucial for immune surveillance and the pathogenesis of various disorders including common inflammatory diseases such as atopic dermatitis or psoriasis, but also malignancies such as cutaneous T cell lymphomas. Cutaneous recruitment of lymphocytes is a highly complex process that involves extravasation, migration through the dermal connective tissue, and eventually, localization to the epidermis. An intertwined network of cytokines and chemokines provides the road signs for leukocyte migration, while various adhesion receptors orchestrate the dynamic events of cell-cell and cell-substrate interactions resulting in cutaneous localization of T cells. Selectively targeting the functions of molecules involved in this interplay promises exciting new therapeutic options for treating inflammatory skin disorders.

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation.

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.