Dany Nassar

Calpain activity is essential in skin wound healing and contributes to scar formation.

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

Pregnancy Promotes Melanoma Metastasis through Enhanced Lymphangiogenesis

The relationships of pregnancy and melanoma have been debatable. Our aim was to assess the influence of gestation on the course of melanoma in a classic murine model of tumor progression and in women. B16 mouse melanoma cells were injected in nonpregnant or pregnant mice on day 5 of gestation. Animals were evaluated for tumor progression, metastases, and survival. Tumor sections were analyzed for lymphatic and blood vessel number and relative surface and expression of angiogenic growth factors. Finally, primary melanomas from pregnant and nonpregnant women, matched for age and tumor thickness, were also considered. Tumor growth, metastasis, and mortality were increased in B16-injected pregnant mice. Tumors displayed an increase in intratumoral lymphangiogenesis during gestation. This increased lymphatic angiogenesis was not observed in normal skin during gestation, showing its specificity to the tumor. An analysis of melanoma from pregnant and matched nonpregnant women showed a similar increase in lymphatic vessels. Tumors from pregnant mice had increased expression of vascular endothelial growth factor A at the RNA and protein levels. The increased vascular endothelial growth factor A production by melanoma cells could be reproduced in culture using pregnant mouse serum. In conclusion, pregnancy results in increased lymphangiogenesis and subsequent metastasis. Caution should be applied in the management of patients with advanced-stage melanoma during gestation.

Calpains Contribute to Vascular Repair in Rapidly Progressive Form of Glomerulonephritis: Potential Role of Their Externalization

Objective—Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. Methods and Results—A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. Conclusion—This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.

Fetal microchimerism in skin wound healing.

Skin wound healing is a complex regenerative process involving various cell types. We recently investigated whether fetal microchimeric cells (FMCs) acquired during gestation contribute to maternal wound healing and used fetal microchimerism to investigate the recruitment of distant endothelial progenitor cells in skin wounds. Our study showed that fetal progenitor cells are recruited into maternal wounds and participate in inflammation and angiogenesis. These fetal cells might have beneficial effects in situations of maternal defective healing, and might also modify the adult maternal wound environment toward a scarless fetal-like wound healing.

Re-Epithelialization of Pathological Cutaneous Wounds Is Improved by Local Mineralocorticoid Receptor Antagonism

Impaired cutaneous wound healing is a social burden. It occurs as a consequence of glucocorticoid treatment in several pathologies. Glucocorticoids (GC) bind not only to the glucocorticoid receptor but also to the mineralocorticoid receptor (MR), both expressed by keratinocytes. In addition to its beneficial effects through the glucocorticoid receptor, GC exposure may lead to inappropriate MR occupancy. We hypothesized that dermatological use of MR antagonists (MRA) might be beneficial by overcoming the negative impact of GC treatment on pathological wounds. The potent GC clobetasol, applied as an ointment to mouse skin, or added to cultured human skin explants, induced delayed wound closure and outgrowth of epidermis with reduced proliferation of keratinocytes. Delayed wound re-epithelialization was rescued by local MRA application. Normal skin was unaffected by MRA. The benefit of MR blockade is explained by the increased expression of MR in clobetasol-treated mouse skin. Blockade of the epithelial sodium channel by phenamil also rescued cultured human skin explants from GC-impaired growth of the epidermis. MRA application over post-biopsy wounds of clobetasol-treated skin zones of healthy volunteers (from the Interest of Topical Spironolactones Administration to Prevent Corticoid-induced Epidermal Atrophy clinical trial) also accelerated wound closure. In conclusion, we propose repositioning MRA for cutaneous application to improve delayed wound closure occurring in pathology.

Ccl2/Ccr2 signalling recruits a distinct fetal microchimeric population that rescues delayed maternal wound healing

Foetal microchimeric cells (FMCs) traffic into maternal circulation during pregnancy and persist for decades after delivery. Upon maternal injury, FMCs migrate to affected sites where they participate in tissue healing. However, the specific signals regulating the trafficking of FMCs to injury sites had to be identified. Here we report that, in mice, a subset of FMCs implicated in tissue repair displays CD11b+ CD34+ CD31+ phenotype and highly express C-C chemokine receptor 2 (Ccr2). The Ccr2 ligand chemokine ligand 2 (Ccl2) enhances the recruitment of FMCs to maternal wounds where these cells transdifferentiate into endothelial cells and stimulate angiogenesis through Cxcl1 secretion. Ccl2 administration improves delayed maternal wound healing in pregnant and postpartum mice but never in virgin ones. This role of Ccl2/Ccr2 signalling opens new strategies for tissue repair through natural stem cell therapy, a concept that can be later applied to other types of maternal diseases.

Delayed Healing of Sickle Cell Ulcers Is due to Impaired Angiogenesis and CXCL12 Secretion in Skin Wounds

Leg ulcers are a major complication of sickle cell disease that occur in 2.5-40% of patients. Leg ulcers are responsible for frequent complications because they are often long-lasting and are highly resistant to therapy. Although their occurrence is associated with hyperhemolysis, the mechanisms underlying sickle cell ulcers remain poorly understood. In this study, we show that skin wound healing is severely altered in old SAD sickle cell mice but is normal in young animals, consistent with reports in humans. Alterations of wound healing were associated with impaired blood and lymphatic angiogenesis in the wound beds and poor endothelial progenitor cell mobilization from the bone marrow. CXCL12 secretion by keratinocytes and inflammatory cells was low in the wounds of SAD mice. Local therapy with endothelial progenitor cells or recombinant CXCL12 injections restored wound angiogenesis and rescued the healing defect together with mobilization of circulating endothelial progenitor cells. To our knowledge, this is a previously unreported study of the cellular and molecular mechanisms of sickle cell ulcers in a murine model that provides promising therapeutic perspectives for clinical trials.

Microchimerism in Mouse Pregnancy

Chapter Summary Microchimerism during and following pregnancy in species with hemochorial placentation is common. That is, very small numbers of maternal cells are found in the fetus and persist in the offspring, and very small numbers of fetal cells occur and can persist in the mother. The importance of these cells, which are detectable for decades, remains an enigma clinically. Mouse models support the human clinical findings that microchimeric cell populations differentiate and that pregnancy-engrafted cells are associated with autoimmune lesions, sites of cancer, and tissue repair. Expansion of well-designed experiments of pregnancy-induced microchimerism would aid in understanding the importance of these grafts in clinical practice.

Fetal cells persist in maternal chronic wounds, form blood vessels and enhance healing

Collagen XVII (COL17), a transmembrane collagen, is thought to be involved in keratinocyte adhesion and possibly migration, as COL17 defects disrupt keratinocytebasal lamina adhesion and underlie the disease non-Herlitz junctional epidermolysis bullosa (n-HJEB). COL17 is involved in keratinocyte adhesion and migration. Using siRNA to knockdown COL17 expression in HaCaT cells, we assessed cell characteristics including adhesion, migration and signaling. Control and siRNA transfected keratinocytes showed no difference in adhesion on plastic dishes after incubation for 8 hours in serum-free keratinocyte-growth medium, however when grown on collagen IV alone or BD matrigel (containing collagen IV and laminin isoforms) COL17 defi cient cells showed signifi cantly reduced adhesion compared to controls (P<0.01), and MEK1/2 and MAPK demonstrated reduced phosphorylation in COL17 depleted cells. Furthermore, COL17 defi cient HaCaT cells plated on plastic exhibited reduced motility that was p38MAP kinase dependent (after addition of the p38MAPK inhibitor SB203580). Conversely, keratinocyte adhesion was independent of p38MAPK signaling. Taken together, these results suggest COL17 has signifi cantly wider signaling roles than were previously thought, including the involvement of COL17 in keratinocyte adhesion to collagen IV, in p38MAP kinase-dependent cell migration, and multiple cell signaling events pertaining to MEK1/2 phosphorylation.