Retrovirally mediated overexpression of versican v3 reverses impaired elastogenesis and heightened proliferation exhibited by fibroblasts from Costello syndrome and Hurler disease patients

Abstract

The phenotypic resemblance of patients with Costello syndrome and Hurler disease has been linked to impaired formation of elastic fibers that coincides with elevated cellular proliferation. Impaired elastogenesis in these diseases associates with respective abnormal accumulation of chondroitin sulfate and dermatan sulfate proteoglycans that induce cell surface shedding of elastin-binding protein (EBP) normally required for intracellular chaperoning of tropoelastin and its assembly into elastic fibers. A variant of the chondroitin sulfate proteoglycan versican, V3, which lacks chondroitin sulfate, has recently been shown to stimulate elastic fiber assembly and decrease proliferation when expressed by retroviral transduction in arterial smooth muscle cells. However, the mechanism(s) by which V3 influences this phenotype is not known. We now demonstrate that transduction of skin fibroblasts from Costello syndrome and Hurler disease patients with cDNA to versican V3 completely reverses impaired elastogenesis and restores normal proliferation of these cells. This phenotypic reversal is accompanied by loss of chondroitin sulfate from the cell surface and increased levels of EBP. Versican V3 transduction of skin fibroblasts from GM(1)-gangliosidosis patients, which lack EBP, failed to restore impaired elastogenesis. These results suggest that induction of elastic fiber production by gene transfer of versican V3 in skin fibroblasts is mediated by rescue of the tropoelastin chaperone, EBP.

Figure 1

A: Representative Northern blots illustrating expression of V3 versican mRNA in Costello syndrome, Hurler disease, and GM₁-gangliosidosis fibroblasts cells retrovirally transduced with V3 cDNA (LV) or with empty vector (LX). B: Northern blots probed with 0.9 kb of human tropoelastin cDNA demonstrate that all tested fibroblasts, either nontransduced (NT) or LX- and LV-transduced, express comparable levels of tropoelastin mRNA.

Figure 2

A: Representative photomicrographs of 48-hour-old cultures of human skin fibroblasts. Note that positive immunostaining is FITC (green) with a red nuclear counterstain. Immunostaining indicates that, in contrast to cultures of normal fibroblasts, Costello syndrome fibroblasts, either nontransduced (NT) or LX-transduced, show strong deposition of chondroitin sulfate-containing moieties (CS), including versican 1, in their extracellular matrix. The high expression of chondroitin sulfate and versican 1 coincided with greatly diminished levels of the cell-surface elastin-binding protein (EBP). In contrast, the LV fibroblasts demonstrated lower levels of immunodetectable chondroitin sulfate moieties and versican 1 and strong cell-surface expression of the EBP, with a pattern that resembled normal fibroblasts. B: Similarly, LV but not LX Hurler fibroblasts showed intense immunostaining for EBP. The LX or LV GM₁-gangliosidosis fibroblasts did not reveal immunodetectable EBP. C: Representative autoradiographs showing levels of the radioactive 67-kd EBP protein purified by elastin affinity columns from the fibroblasts metabolically pulsed with [¹⁴C]-serine for 1 hour and than chased for 45 minutes. In contrast to normal fibroblasts, which retain the majority of metabolically labeled EBP after a 45-minute chase, fibroblasts maintained in the presence of exogenous chondroitin sulfate (CS) lose a large portion of this initially synthesized protein. Moreover, LV fibroblasts from Costello syndrome and Hurler disease retain more 67-kd EBP than their LX counterparts. Both LX and LV GM₁-gangliosidosis fibroblasts do not contain any metabolically labeled EBP.

Figure 3

Representative photomicrographs of 7-day-old cultures immunostained with anti-tropoelastin antibody, indicate that normal fibroblasts produced long, branching elastic fibers, whereas normal fibroblasts maintained in the presence of exogenous chondroitin sulfate do not deposit any extracellular elastin. The lack of elastic fibers is also apparent in cultures of Costello syndrome and Hurler disease fibroblasts transduced with empty retroviral vector (LX). In contrast, Costello and Hurler fibroblasts transduced with cDNA encoding V3 versican (LV) produce elastic fibers that are similar to those deposited in cultures of normal fibroblasts. The EBP-deficient fibroblasts derived from GM₁-gangliosidosis patients did not display any elastic fibers in the newly produced extracellular matrix regardless of LX or LV transduction.

Figure 4

Morphometric analysis of immunostained components of extracellular matrix in 7-day-old cultures of normal and Costello syndrome fibroblasts (A), and in cultures of Hurler disease (B) and GM₁-gangliosidosis fibroblasts (C). The nontransduced (NT) and empty vector-transduced (LX) Costello and Hurler fibroblasts deposit only negligible amounts of immunodetectable extracellular elastin. Amounts of chondroitin sulfate produced by these fibroblasts significantly exceed those present in cultures of normal fibroblasts. In contrast, both V3-transduced (LV) Costello and Hurler, but not GM₁-gangliosidosis-derived fibroblasts, demonstrate significantly higher rates of immunodetectable elastin in their ECM, but lower accumulation of chondroitin sulfate. Deposition of fibrillin 1 and MAGP (marking microfibrillar scaffold) in all tested cultures was not affected by LX or LV transduction. In each analyzed group, 50 low-power fields (×20) from three separate cultures (per independent patients) were analyzed and the area occupied by the particular immunodetectable component quantified. The abundance of each component was then expressed as a percentage of the entire analyzed field (mean ± SD), and results from cultures of fibroblasts from Costello syndrome and Hurlers disease were statistically compared with those in cultures of normal skin fibroblasts (*, P < 0.001).

Figure 5

A: Quantitative analysis of [³H]-valine-labeled immunoprecipitable tropoelastin indicates that normal (patient 4184), Costello syndrome (patients 12,368 and 12,195), Hurler disease (patients 8180 and 8339), and GM₁-gangliosidosis (patient 4032) fibroblasts synthesize comparable amounts of total metabolically labeled tropoelastin. B: In contrast to normal fibroblasts, all nontransduced (NT) and LX fibroblasts retain the majority of their metabolically labeled tropoelastin intracellularly. Consistently, those fibroblasts demonstrate lower than normal levels of immunoprecipitable tropoelastin from conditioned media (C) and incorporate significantly less [³H]-valine into extracellular insoluble elastin than normal fibroblasts (D). In contrast, cultures of both Costello syndrome and Hurler disease fibroblasts transduced with the V3 versican gene (LV) demonstrate much lower levels of intracellular tropoelastin and significantly higher levels of insoluble elastin than their NT and LX counterparts. LV transduction of GM₁-gangliosidosis fibroblasts did not improve their ability to secrete newly synthesized tropoelastin and deposit insoluble elastin.

Figure 6

A: Assessment of [³H]-thymidine incorporation into 3-day-old cultures (initially densely seeded at 100,000 cells/dish to reach immediate confluency and deposition of elastin-containing ECM). Results indicated the V3 transduction of Costello and Hurler, but not GM₁-gangliosidosis fibroblasts, causes a significant decrease in their proliferation rate. B: Estimation of cell number and total DNA assayed in dense cultures (initially plated at 100,000 cells/dish). The NT and LX Costello and Hurler fibroblasts exhibit significantly higher growth rates. In contrast, growth curves of their LV counterparts are similar to normal fibroblasts.

Figure 7

Incorporation of [³H]-valine into NaOH-insoluble elastin (A) and incorporation of [³H]-thymidine (B) in subconfluent 3-day-old cultures of Costello syndrome and Hurler disease fibroblasts (initially sparsely seeded at 20,000 cells/dish). Both NT and LX Costello and Hurler fibroblasts kept in subconfluent cultures do not deposit significant amounts of elastin and demonstrate only slightly higher than normal proliferation rates. The proliferation of LV fibroblasts kept in subconfluent cultures does not differ from their NT and LX counterparts.

Figure 8

A:Quantitative analysis of [³H]-valine-labeled insoluble elastin indicates that treatment with inhibitor of lysyl oxidase, β-amino-prioprio-nitrile (BAPN) caused radical inhibition in deposition of insoluble elastin in densely plated (100,000 cells/dish), 7-day-old cultures of normal fibroblasts and V3-transduced (LV) Costello fibroblasts. Treatment with BAPN further deteriorates low elastin deposition in cultures of NT and LX Costello fibroblasts. B: Assessment of [³H]-thymidine incorporation in parallel 7-day-old cultures demonstrates that V3-transduction of (LV) did not reduce heightened proliferation of Costello fibroblasts cultured in the presence of BAPN, which prevents the efficient deposition of insoluble elastin. Interestingly, inhibition of insoluble elastin deposition in cultures of normal fibroblasts also caused an increase in their proliferation rate.