Fibroblast growth factors: key players in regeneration and tissue repair.

Tissue injury initiates a complex repair process, which in some organisms can lead to the complete regeneration of a tissue. In mammals, however, the repair of most organs is imperfect and results in scar formation. Both regeneration and repair are orchestrated by a highly coordinated interplay of different growth factors and cytokines. Among the key players are the fibroblast growth factors (FGFs), which control the migration, proliferation, differentiation and survival of different cell types. In addition, FGFs influence the expression of other factors involved in the regenerative response. Here, we summarize current knowledge on the roles of endogenous FGFs in regeneration and repair in different organisms and in different tissues and organs. Gaining a better understanding of these FGF activities is important for appropriate modulation of FGF signaling after injury to prevent impaired healing and to promote organ regeneration in humans.

The signaling adaptor Eps8 is an essential actin capping protein for dendritic cell migration.

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.

EndMT contributes to the onset and progression of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-ß and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-ß and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.

Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice.

Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of ß-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a ß-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-ß/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate ß-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Accumulation and activation of epidermal γδ T cells in a mouse model of chronic dermatitis is not required for the inflammatory phenotype.

Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5-R1/R2 mice) develop an AD-like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5-R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il-2 and Il-7 and the stress-induced proteins Rae-1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5-R1/-R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5-R1/R2 mice, possibly due to partial compensation by αß T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier.

CCM1 regulates vascular-lumen organization by inducing endothelial polarity.

Little is known about the molecular mechanisms that regulate the organization of vascular lumen. In this paper we show that lumen formation correlates with endothelial polarization. Adherens junctions (AJs) and VE-cadherin (VEC, encoded by CDH5) are required for endothelial apicobasal polarity in vitro and during embryonic development. Silencing of CDH5 gene expression leads to abrogation of endothelial polarity accompanied by strong alterations in lumenal structure. VEC co-distributes with members of the Par polarity complex (Par3 and PKCzeta) and is needed for activation of PKCzeta. CCM1 is encoded by the CCM1 gene, which is mutated in 60% of patients affected by cerebral cavernous malformation (CCM). The protein interacts with VEC and directs AJ organization and AJ association with the polarity complex, both in cell-culture models and in human CCM1 lesions. Both VEC and CCM1 control Rap1 concentration at cell-cell junctions. We propose that VEC, CCM1 and Rap1 form a signaling complex. In the absence of any of these proteins, AJs are dismantled, cell polarity is lost and vascular lumenal structure is severely altered.

The functional role of cell adhesion molecules in tumor angiogenesis.

Cell adhesion molecules (CAMs) are cell surface glycoproteins that mediate the physical interactions between adjacent cells and between cells and the surrounding extracellular matrix. CAMs belong to different protein families, depending on their structural and functional properties. Furthermore, the expression of certain CAMs under physiological conditions is restricted to specific cell types. Besides playing a key homeostatic role in maintaining the architecture of quiescent tissues, CAMs have also to adapt to the microenvironmental changes that occur during certain physiological and pathological processes. This is best exemplified by cancer vascularization, where the expression and function of vascular CAMs are dynamically regulated in response to tissue alterations induced by tumor growth as well as by changes in the surrounding stroma. This enables endothelial cells (ECs) to leave the quiescent state and re-enter the angiogenic cascade. The latter is a multistep process carried out by different types of specialized ECs. This review describes the actual or supposed function of the various CAM subsets in the sequential series of events that underlie vascular changes during tumor angiogenesis. Notably, elucidating the mechanism of action of endothelial CAMs in cancer vasculature is expected to open new therapeutic avenues aimed at interfering with tumor growth and dissemination.

Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice.

FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.

Antagonism of interferon signaling by fibroblast growth factors promotes viral replication.

Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross-talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon-stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF-mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV-1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV-1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections.

KLF4 is a key determinant in the development and progression of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFß/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFß/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFß/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.