Severe systemic fibrosis is an universal observation in postmortem and biopsy specimens of ALMS patients and may result in early organ failure and death 
. Whether the fibrotic damage is a direct result of mutations in the ALMS1
gene or, alternatively, a secondary response to cellular insult due to the loss of ALMS1 function is unknown. However, while hepatic and renal fibrosis could possibly be explained by the metabolic alterations present in ALMS patients, such as obesity and type 2 diabetes, it is difficult to identify a trigger insult causing fibrosis in heart, lung, bladder, testis and ovary.
Fibrosis is often considered a fibroproliferative disorder in which the uncontrolled proliferation of activated fibroblasts and the excessive production of ECM resulted in a functional impairment of affected organs 
We did not observe an enhanced proliferative capacity in ALMS fibroblasts. On the contrary, ALMS1 mutations in the majority of our patients are associated with an increase in cell cycle length and in all patients with a down-regulation of genes directly involved in cell cycle progression, replication and centrosome-kinetocore assembly. Fibroblasts obtained from PT4 displayed a normal cell cycle length and it is noteworthy that only PT4 carried a mutation in exon 16 of ALMS1, thus he could preserve some ALMS function in hemizygosity. Additional studies regarding the different disease-causing variants will help us to gain a better understanding of the phenotypic variability observed among ALMS patients.
The localization of ALMS1 to centrosomes and ciliary basal bodies 
suggests a role in the regulation of cell cycle progression. Primary cilia are regarded as postmitotic structures of quiescent cells 
and recently were proposed to be involved in the control of cellular growth. ALMS overlaps clinical phenotypes of other ciliopathies, as Barbet-Biedl Syndrome (BBS), and a role for BBS proteins in microtubule organization and cell cycle progression has been hypothesized 
. The increased doubling time we observed in the majority of ALMS fibroblasts is the first evidence which links ALMS1 function to the regulation of cell cycle progression.
We showed the up-regulation of several genes coding for ECM components and regulators associated with increased collagen synthesis in ALMS fibroblasts. The enhanced intracellular complexity and the active exocytic dynamic assayed by our morphological evaluations similarly suggest a constitutive activated phenotype for dermal ALMS fibroblasts. Furthermore, control fibroblasts became able to express the same COL1A1 levels as ALMS fibroblasts only upon 48 hour-treatment with TGF-β. Additionally, ALMS fibroblasts remain responsive to TGF-β and the stimulation further increased their COL1A1 expression more efficiently than controls.
Array experiments showed the mRNA modulation in ALMS fibroblasts of four out of the six matricellular growth factors belongs to the CCN family: CTGF
, all of which function as adaptor molecules connecting the cell surface to the ECM. Constitutive over-expression of CTGF
has been linked with systemic sclerosis, keloids and other fibrotic skin disorders 
and the up-regulation of WISP1
, a WNT signaling pathway target, has been associated with human idiopathic pulmonary fibrosis 
. We also observed a marked up-regulation of POSTN
, a protein involved in proper ECM synthesis 
and in the hypertrophic response following myocardial infarction 
. The strong periostin overexpression we observed together with the risk described for developing cardiomyopathy in ALMS patients 
is particularly suggestive of a role of periostin in cardiac remodeling in ALMS.
Taken together all these observations show that ECM deposition is a dominant pathological mechanism of ALMS fibrosis, is constitutively present in mutated fibroblasts, and could be enhanced by the presence of a profibrotic environment.
The 3D-culture experiments showed that ALMS fibroblasts display an inability to migrate into the inner spaces of the scaffold, appearing strongly anchored to adjacent cells. This result is in agreement with expression data indicating the up-regulation of genes involved in focal adhesion structures and suggesting a closer cell-matrix association. Furthermore, our morphological analysis showed the presence of cytoskeletal structures preferentially lined up to the nuclear polarity in ALMS fibroblasts compared to controls. The migration and the orientation of fibroblasts with respect to the collagen fibers are both essential steps in normal tissue repair 
. Thus, the lack of ALMS1 function could alter the cell polarity and the efficient migration contributing to the generation of fibrosis.
Resistance to apoptosis has been described in fibroblasts isolated from fibrotic lesions in patients with pulmonary fibrosis 
or with keloids 
. Our microarray analysis suggests that ALMS1
-mutated fibroblasts could possess some alterations in genes involved in the apoptotic pathway regulation and partially in the control of stress-induced cell death. Moreover the treatment with some apoptotic inductors (THAP, C2
-C and CX) is more efficient in controls than in ALMS fibroblasts, indicating that a specific apoptotic pathway could be altered in mutated cells. These results suggest that ALMS fibroblasts display a “prosurvival phenotype” that could play a role in the generation of fibrosis interfering with the control of the fibroproliferative response.
Dermal ALMS fibroblasts display a constitutive activation of an α-SMA-myofibroblast like phenotype, indicating autonomous, signal-independent alterations, directly due to mutations in ALMS1 gene. They divide more slowly and are less susceptible to apoptosis, thus continue to proliferate in an uncontrolled manner and produce an excess of ECM, resulting in fibrosis. Moreover ALMS fibroblasts keep themselves responsive to profibrotic inductors contributing to the perpetuation of fibrosis ().
Model of fibrosis in Alström Syndrome.
In conclusion, we show that fibrosis in ALMS is a primary defect due to ALMS1 mutations leading to the fibrotic phenotype described in ALMS patients. Moreover, ALMS1 is a multifunctional protein, with roles in cell cycle progression, migration, apoptosis and ECM production. Further studies investigating the involvement of ALMS1 in both intra- and extra-cellular events will be helpful in unraveling the pathophysiology of ALMS to facilitate the identification of key therapeutic targets.