Paclitaxel modulates TGFbeta signaling in scleroderma skin grafts in immunodeficient mice

Abstract

Background: Systemic sclerosis (SSc) is characterized by excessive fibrosis and obliterative vascular lesions. Abnormal TGFbeta activation is implicated in the pathogenesis of SSc. Aberrant TGFbeta/Smad signaling can be controlled by stabilization of microtubules with paclitaxel.

Methods and findings: SSc and healthy human skin biopsies were incubated in the presence or absence of paclitaxel followed by transplantation into severe combined immunodeficient mice. TGFbeta signaling, fibrosis, and neovessel formation were evaluated by quantitative RT-PCR and immunohistochemical staining. Paclitaxel markedly suppressed Smad2 and Smad3 phosphorylation and collagen deposition in SSc grafts. As a result, the autonomous maintenance/reconstitution of the SSc phenotype was prevented. Remarkably, SSc grafts showed a 2-fold increase in neovessel formation relative to normal grafts, regardless of paclitaxel treatment. Angiogenesis in SSc grafts was associated with a substantial increase in mouse PECAM-1 expression, indicating the mouse origin of the neovascular cells.

Conclusion: Low-dose paclitaxel can significantly suppress TGFbeta/Smad activity and lessen fibrosis in SCID mice. Transplantation of SSc skin into SCID mice elicits a strong angiogenesis-an effect not affected by paclitaxel. Although prolonged chemotherapy with paclitaxel at higher doses is associated with pro-fibrotic and anti-angiogenic changes, the findings described here indicate that low-dose paclitaxel may have therapeutic benefits for SSc via modulating TGFbeta signaling.

Figure 1. Paclitaxel Treatment Suppresses Smad2 Phosphorylation

Strong nuclear staining for phospho-Smad2 (A) and total Smad2 (B) is observed in an SSc skin graft. Smad2 phosphorylation (C) is rare in normal graft, which expresses abundant Smad2 (D). Smad2 phosphorylation is suppressed with paclitaxel (Taxol) treatment (E), without affecting total Smad2 (F) in SSc skin graft. Tx, transplantation.

Figure 2. Paclitaxel Decreases COLA2 mRNA Expression in SSc Grafts

TRT-PCR analysis shows that the expression of COL1A2 is reduced 4.5-fold with paclitaxel (Taxol) treatment in SSC grafts, reaching a level equivalent to that of normal skin grafts. By contrast, paclitaxel has no effects on COL1A2 expression in normal skin grafts.

Figure 3. Paclitaxel Decreases Collagen Deposition in SSc Grafts

VVM staining shows abundant, thick collagen bundles in the SSc graft (A). The amount of collagen is markedly reduced, and the collagen fibers become finer with paclitaxel (Taxol) treatment (B), comparable to that seen in the normal skin graft (C). Similarly, IHC staining demonstrates intense collagen-1 staining in the SSc graft (D), which is substantially decreased with paclitaxel treatment (E) to a level similar to that seen in the normal skin graft (F).

Figure 4. SSc Skin Grafting Stimulates Angiogenesis in SCID Mice

Macroscopic analysis of the vessels in the underside of normal (A and B) and SSc (C and D) skin grafts reveals more pronounced angiogenesis in SSc grafts; paclitaxel (Taxol) treatment has no effect on angiogenesis at the macroscopic level (B and D versus A and C). Microscopic analysis shows that the number of vessels in three high-power fields of the SSc grafts is greater than that of normal skin grafts; paclitaxel has no effect on angiogenesis at the microscopic level (E). TRT-PCR for mouse PECAM-1 demonstrates a substantial increase in mouse PECAM-1 mRNA expression in SSc and normal skin grafts regardless of paclitaxel treatment; the amplitude of the increase is significantly greater in SSc than in normal skin grafts (F). Human PECAM-1 is expressed in much lower levels in SSc than in normal skin tissues, indicative of vascular deficiency in SSc; the lack of difference in the expression level of human PECAM-1 in SSc and normal skin tissues before and after transplantation indicates that the neovascular cells are derived from the recipients (G).