p21cip/WAF is a key regulator of long-term radiation damage in mesenchyme-derived tissues.

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition, we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated >200-fold in irradiated tissues. Loss of p21 function resulted in a >4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage, including 6-fold less scarring, 40% increased growth potential, and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (P<0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation, 4-fold greater proliferation rate, and nearly 7-fold lower senescence as compared to WT MSCs (P<0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition, p21 is a critical regulator of MSC proliferation, differentiation, and senescence both at baseline and in response to radiation.

Radiation therapy causes loss of dermal lymphatic vessels and interferes with lymphatic function by TGF-beta1-mediated tissue fibrosis.

Although radiation therapy is a major risk factor for the development of lymphedema following lymphadenectomy, the mechanisms responsible for this effect remain unknown. The purpose of this study was therefore to determine the effects of radiation on lymphatic endothelial cells (LECs) and lymphatic function. The tails of wild-type or acid sphingomyelinase (ASM)-deficient mice were treated with 0, 15, or 30 Gy of radiation and then analyzed for LEC apoptosis and lymphatic function at various time points. To analyze the effects of radiation fibrosis on lymphatic function, we determined the effects of transforming growth factor (TGF)-beta1 blockade after radiation in vivo. Finally, we determined the effects of radiation and exogenous TGF-beta1 on LECs in vitro. Radiation caused mild edema that resolved after 12-24 wk. Interestingly, despite resolution of tail edema, irradiated animals displayed persistent lymphatic dysfunction. Radiation caused loss of capillary lymphatics and was associated with a dose-dependent increase in LEC apoptosis. ASM-/- mice had significantly less LEC apoptosis; however, this finding did not translate to improved lymphatic function at later time points. Short-term blockade of TGF-beta1 function after radiation markedly decreased tissue fibrosis and significantly improved lymphatic function but did not alter LEC apoptosis. Radiation therapy decreases lymphatic reserve by causing depletion of lymphatic vessels and LECs as well as promoting soft tissue fibrosis. Short-term inhibition of TGF-beta1 activity following radiation improves lymphatic function and is associated with decreased soft tissue fibrosis. ASM deficiency confers LEC protection from radiation-induced apoptosis but does not prevent lymphatic dysfunction.

Fibrosis is a key inhibitor of lymphatic regeneration.

BACKGROUND: Lymphedema is a common debilitating sequela of lymph node dissection. Although numerous clinical studies suggest that factors that lead to fibrosis are associated with the development of lymphedema, this relationship has not been proven. The purpose of these experiments was therefore to evaluate lymphatic regeneration in the setting of variable soft-tissue fibrosis. METHODS: A section of mouse tail skin including the capillary and collecting lymphatics was excised. Experimental animals (n = 20) were treated with topical collagen type I gel and a moist dressing, whereas control animals (n = 20) underwent excision followed by moist dressing alone. Fibrosis, acute lymphedema, lymphatic function, gene expression, lymphatic endothelial cell proliferation, and lymphatic fibrosis were evaluated at various time points. RESULTS: Collagen gel treatment significantly decreased fibrosis, with an attendant decrease in acute lymphedema and improved lymphatic function. Tails treated with collagen gel demonstrated greater numbers of lymphatic vessels, more normal lymphatic architecture, and more proliferating lymphatic endothelial cells. These findings appeared to be independent of vascular endothelial growth factor C expression. Decreased fibrosis was associated with a significant decrease in the expression of extracellular matrix components. Finally, decreased soft-tissue fibrosis was associated with a significant decrease in lymphatic fibrosis as evidenced by the number of lymphatic endothelial cells that coexpressed lymphatic and fibroblast markers. CONCLUSIONS: Soft-tissue fibrosis is associated with impairment in lymphatic regeneration and lymphatic function. These defects occur as a consequence of impaired lymphatic endothelial cell proliferation, abnormal lymphatic microarchitecture, and lymphatic fibrosis. Inhibition of fibrosis using a simple topical dressing can markedly accelerate lymphatic repair and promote regeneration of normal capillary lymphatics.