Background Scleroderma fibroblasts are characterized by elevated adhesive signaling. PTEN, a phosphatase that suppresses adhesive signaling, expression is reduced in scleroderma dermal fibroblasts. Previously, we showed that loss of PTEN expression in dermal mouse fibroblasts results in skin fibrosis associated with an elevated expression of the fibrotic marker CTGF/CCN2. .
Objectives Our aim is to whether (a) loss of PTEN expression in fibroblasts also results in lung fibrosis ands (b) whether CCN2 mediates lung fibrosis caused by loss of PTEN.
Methods A cre/lox system is used to delete PTEN and/or CCN2 in fibroblasts. Mice expressing a tamoxifen-dependent cre under the control of a fibroblast specific (COL1A2) promoter were generated that were homozygous for a loxP-PTEN allele and/or a loxP-CCN2 allele. Mice were injected with corn oil (to generate control mice) or tamoxifen (to delete PTEN and/or CCN2). Forty five days post-cessation of tamoxifen injection, lungs were harvested and subjected to histological and biochemical analyses to assess the effect of loss of PTEN and/or CCN2 on lung fibrogenesis (ie, Hematoxylin and eosin and trichrome stains, hydroxyproline/proline analysis, real time polymerase chain analysis, and immunohistological analyses with anti-type I collagen, anti-p-Akt, anti-CCN2, anti-phospho-Smad3 and anti-a-smooth muscle actin antibodies). Blood vessels were detected using anti-CD31 antibodies. Anti-NG2 antibodies were used to detect expansion of microvascular pericytes.
Results Loss of PTEN expression resulted in lung fibrosis around the blood vessels. Loss of PTEN resulted in collagen and a-smooth muscle actin expression and deposition as visualized by immunohistochemical analysis (N=6, p<0.05) and real time PCR analysis (N=3, p<0.05), but not in PTEN/CCN2 double knockout animals. Smad3 and Akt phosphorylation were elevated in PTEN knockout mice; loss of CCN2 rescued elevated Smad3 but not Akt phosphorylation (N=6, p<0.05). Double labeling analysis revealed that a-smooth muscle actin-postive cells were also NG2-positive, suggesting that these cells appeared to arise from a pericyte or pericyte-like population (N-6, p<0.05).
Conclusions Loss of PTEN in fibroblasts results in lung fibrosis, in a fashion dependent on CCN2. Thus mice deficient in PTEN may represent a novel model for certain features of scleroderma. These results moreover indicate that elevated adhesive signaling (due to loss of PTEN expression) is sufficient for fibrogenesis and that CCN2 may represent a common downstream mediator of fibrosis, possibly due to its ability to promote expansion of a pericyte or pericyte-like cell population.
Disclosure of Interest None Declared
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