Table 1.
Genetics and clinical characteristics of four ALMS patients studied.
Figure 1.
Size, shape and ultrastructural evaluation of ALMS fibroblasts.
(a) Fibroblasts of healthy control (C3) and (b) ALMS patient (PT3) were grown on standard tissue culture (2D cultures) coverslips and stained with hematoxylin-eosin (magnification 20×). (c) Cell length of about 40 fibroblasts per subject was quantified by measuring the longitudinal cell length and was plotted as mean (horizontal line), maximal and minimal length values (vertical line). **P<0.01 ALMS fibroblast length mean versus controls length mean. (d) The area covered by fibroblast cells during exponential growth was estimated by cell counting in Bürker chambers, after 0.2% trypan blue staining. Results are reported as mean ± SEM. *P<0.05. ALMS fibroblasts were compared with controls by flow cytometric analysis: (e) the resulting forward (FSC) and (f) side light scatter (SSC) mean intensity values are shown for each subject. Controls: C1, black triangle; C2, black star; C3, black diamond; Patients: PT1, white circle; PT2, white square; PT3, white triangle; PT4, white diamond (see also Table 1). Panel g reports the SSC distribution of fibroblasts from C1 (black line) and PT1 (grey line) showing the highly significant shift (***P<0.001, as determined by the Kolmogorov-Smirnov analysis according to the Macintosh CELLQuest software user's guide, Becton Dickinson). (h) Fibroblasts of healthy control (C2) and (i) ALMS patient (PT2) were cultured in HYAFF-11™ scaffolds (3D-cultures) and stained with hematoxylin-eosin (magnification 5×; * biomaterial scaffolds; → fibroblast cells). Transmission electron microscopy was performed in healthy control (C2) (l–n–p) and ALMS patient (PT2) (m–o–q) on 3D-cultured cells. Control fibroblasts showed a normal phenotype, with typical bipolar morphology (l), cytoplasm rich in perpendicular oriented microfilaments (n, ring), the presence of normal pinocytic vesicles (l arrow and p) and probably collagen fibers in the extracellular space (p, ring). In contrast, ALMS fibroblasts appeared as elongated cells tightly adherent to each other and showed well-defined long cytoplasmic extensions (m). Microfilaments (o, ring) were arranged in a unique direction, parallel to the long axis of the cells. A large amount of exocytic vesicles and probably collagen fibrils (q, arrow and ring, respectively) suggest active secretion. Magnification: l–m = 5000×; n–o = 25000×; p = 40000×; q = 30000× (see also Figure S3 and S4).
Figure 2.
Distribution of differentially expressed genes in ALMS fibroblasts.
Fibroblast transcriptional profiles were analyzed by whole genome expression experiments using RNA isolated from healthy controls and ALMS fibroblasts. (a) Pie chart shows the percentage of differentially expressed genes (n = 560) in ALMS fibroblasts clustered in main categories: cell cycle (n = 90), ECM components/fibrosis regulation (n = 37), cellular adhesion/motility (n = 24) and apoptosis (n = 18). (b) Bar chart displays the distribution of up- (black) and down- (grey) regulated genes for each identified group with the number of corresponding genes. A detailed description of the whole genome expression analysis is given in Table S2.
Table 2.
Gene Ontology distribution of differentially expressed genes in microarray analysis.
Figure 3.
Increased mRNA expression and deposition of ECM components in ALMS fibroblasts.
(a) POSTN, ACTA2, COL1A1, COL3A1, COL4A1, COL5A1, COL5A2, COL8A1, COL11A1, COL12A1, COL15A1 transcripts were quantified by qPCR and normalized to HMBS mRNA content in control (CONTROLS) and ALMS fibroblasts (ALMS). Results are reported as mean values ± SEM and are expressed as fold change with respect to controls, arbitrarily set as 1 for each of analyzed transcript. *P<0.05, ** P<0.01, *** P<0.001 ALMS fibroblasts versus controls (see also Figure S6). (b) The fold change observed between ALMS and control fibroblasts in microarray analysis was plotted against the fold change measured by qPCR for ACTA2 and COLs transcript. A strong correlation between the two methods was observed. Pearson coefficient = 0.8. (c) Fibroblast collagen protein synthesis and release were determined by [3H]-proline incorporation, and expressed as counts per minute normalized to DNA content. *P<0.05, ***P<0.001 ALMS fibroblasts versus controls. Black bars correspond to control and white bars to ALMS fibroblasts.
Figure 4.
ALMS fibroblasts display a longer cell cycle than controls.
Cell cycle length of control (C1–C3) and ALMS fibroblasts (PT1–PT4) was estimated by culture experiments in 10% FBS SM (white symbols), in 2% FBS SM (black symbols), and upon serum deprivation for 48 hour, followed by culture in 10% FBS SM (grey symbols) by comparing the number of viable cells at 0 hours and at 72 hours of culture, using CellTiter-Glo® Luminescent Cell Viability Assay (Promega). The short dash line indicates the mean value of cell cycle length of controls in all growth conditions analyzed, while the dotted lines represent the mean value ± one SD.
Figure 5.
ALMS fibroblasts are resistant to cell death induced by apoptotic stimuli.
(a) Fibroblasts of healthy controls and ALMS patients were stimulated with thapsigargin (THAP, 100 nM for 48 hours), C2-ceramide (C2-C, 100 µM for 24 hours), cycloheximide (CX, 100 µg/ml for 24 hours), staurosporine (STP, 100 nM for 48 hours) and TNF-α (100 ng/ml for 48 hours) in 10% FBS SM. The % of viable cells was determined by MTT assay and shown as mean values ± SEM with respect to unstimulated cells (−) indicated as 100%. *P<0.05, ***P<0.001 cell viability of ALMS fibroblasts versus controls. (b) Control (C1) and ALMS fibroblast (PT4) were treated with THAP (100 nM for 48 hours), stained with PI and analyzed by flow cytometry. Results are presented as histograms of cell cycle phase distribution and the reported % represents the increase in sub-G1/G0 population (M1 region). (c) Control (C1) and ALMS fibroblast (PT1) were grown on glass coverslips and stimulated with CX (100 µg/ml for 24 hours) and THAP (100 nM for 48 hours). Cells were fixed and stained for fluorescence in situ DNA end-labelling (TUNEL) (green stain); nuclei were counter-stained with DAPI (blue stain) (magnification = 20×). (d) Control (C1) and ALMS fibroblast (PT1) were treated with THAP (100 nM for 48 and 72 hours). Cells were labelled by TUNEL and PI and analyzed by flow cytometry. Each cytogram shows the TUNEL positivity (ordinate) with respect to DNA content (abscissa), upon 48 and 72 hours of THAP-treatment, respectively. A representative control and patient are reported in panel b, c and d; see also Figure S9 and S10.
Figure 6.
Model of fibrosis in Alström Syndrome.
Fibroblasts carrying ALMS1 mutations display an elongated shape, proliferate slowly, but are still responsive to pro-fibrotic factors and resistant to cell-death stimuli, suggesting that their proliferation is not controlled by apoptosis. ALMS1 mutated fibroblasts persist, continue to proliferate and to synthesize and secrete high levels of ECM, responsible for progressively remodeling and destroying normal tissue architecture, resulting in fibrosis. The microenvironment could be characterized by an excess of mediators enhancing a cellular pro-fibrotic phenotype, that together with inflammatory reactions could stimulate the fibrosis in an autocrine loop.