Picroliv Accelerates Epithelialization and Angiogenesis in Rat Wounds

Abstract

Tissue repair and wound healing are complex processes that involve inflammation, granulation and tissue remodeling. Angiogenesis plays a central role in wound healing. Earlier, we have shown that picroliv, a natural product obtained from the roots of Picrorhiza kurrooa, up-regulates the expression of vascular endothelial growth factor in human umbilical vein endothelial cells and of insulin-like growth factor in rats during hypoxia. In the present study, we have investigated the effect of picroliv in an ex vivo rat aorta ring model of angiogenesis. Picroliv enhanced the sprouting and migration of endothelial cells. We also investigated punch wound healing on days 4 and 7 after wounding by histology, morphometry and collagenization. The data showed improved re-epithelialization, neovascularization and migration of various cells such as endothelial, dermal myofibroblasts and fibroblasts into the wound bed after picroliv treatment. Immunohistochemical localization showed increased VEGF and alpha smooth muscle actin staining consistent with an increased number of microvessels in granulation tissue. These findings suggest that picroliv could be developed as a therapeutic angiogenic agent for the restoration of the blood supply in diseases involving inadequate blood supply such as limb ischemia, ischemic myocardium and wound healing.

Key words

Picroliv - angiogenesis - wound healing - fibroblast

References

• 1 Martin P, Hopkinson-Woolley J, McCluskey J. Growth factors and cutaneous wound repair.  Prog Growth Factor Res. 1992;  4 25-44.
• 2 Martin P. Wound healing - aiming for perfect skin regeneration.  Science. 1997;  276 75-81.
• 3 Singer A J, Clark R A. Cutaneous wound healing.  N Engl J Med. 1999;  341 738-46.
• 4 Hansen L A, Alexander N, Hogan M E, Sundberg J P, Dlugosz A, Threadgill D W. et al . Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.  Am J Pathol. 1997;  150 1959-75.
• 5 Miller D L, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice.  Mol Cell Biol. 2000;  20 2260-8.
• 6 Gallucci R M, Simeonova P P, Matheson J M, Kommineni C, Guriel J L, Sugawara T. et al . Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.  FASEB J. 2000;  14 2525-31.
• 7 Howdieshell T R, Callaway D, Webb W L, Gaines M D, Procter  Jr. C D, Sathyanarayana , Pollock J S, Brock T L, McNeil P L. Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation.  J Surg Res. 2001;  96 173-82.
• 8 Rothe M, Falanga V. Growth factors. Their biology and promise in dermatologic diseases and tissue repair.  Arch Dermatol. 1989;  125 1390-8.
• 9 Mustoe T A, Pierce G F, Morishima C, Deuel T F. Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model.  J Clin Invest. 1991;  87 694-703.
• 10 Smith S K. Angiogenesis, vascular endothelial growth factor and the endometrium.  Hum Reprod Update. 1998;  4 509-19.
• 11 Nissen N N, Polverini P J, Koch A E, Volin M V, Gamelli R L, DiPietro L A. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing.  Am J Pathol. 1998;  152 1445-52.
• 12 Isner J M, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T. et al . Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb.  Lancet. 1996;  348 370-4.
• 13 Chander R, Kapoor N K, Dhawan B N. Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions.  Biochem Pharmacol. 1992;  44 180-3.
• 14 Rastogi R, Saksena S, Garg N K, Kapoor N K, Agarwal D P, Dhawan B N. Picroliv protects against alcohol-induced chronic hepatotoxicity in rats.  Planta Med. 1996;  62 283-5.
• 15 Seth P, Sundar S V, Seth R K, Sidhu G S, Sharma S C, Kulshreshtha D K. et al . Picroliv modulates antioxidant status and down-regulates AP1 transcription factor after hemorrhage and resuscitation.  Shock. 2003;  19 169-75.
• 16 Gaddipati J P, Mani H, Banaudha K K, Sharma S K, Kulshreshtha D K, Maheshwari R K. Picroliv modulates the expression of insulin-like growth factor (IGF)-I, IGF-II and IGF-I receptor during hypoxia in rats.  Cell Mol Life Sci. 1999;  56 348-55.
• 17 Gaddipati J P, Madhavan S, Sidhu G S, Singh A K, Seth P, Maheshwari R K. Picroliv - a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation.  Mol Cell Biochem. 1999;  194 271-81.
• 18 Singh A K, Mani H, Seth P, Gaddipati J P, Kumari R, Banuadha K K. et al . Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury.  Eur J Pharmacol. 2000;  395 229-39.
• 19 Seth P, Kumari R, Madhavan S, Singh A K, Mani H, Banaudha K K. et al . Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv.  Biochem Pharmacol. 2000;  59 1315-22.
• 20 Aswal B S, Chander R, Chatterji S K, Dhawan B N, Dwivedi Y, Garg N K. et al .Process for the preparation and composition of a fraction containing picroside1 and kutkoside. US Patent 5 145 955. 1992.
• 21 Sidhu G S, Mani H, Gaddipati J P, Singh A K, Seth P, Banaudha K K. et al . Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice.  Wound Repair Regen. 1999;  7 362-74.
• 22 Clark R A. Biology of dermal wound repair.  Dermatol Clin. 1993;  11 647-66.
• 23 Risau W. Angiogenic growth factors.  Prog Growth Factor Res. 1990;  2 71-9.
• 24 Benjamin L E, Keshet E. Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.  Proc Natl Acad Sci USA. 1997;  94 8761-6.
• 25 Nicosia R F, Lin Y J, Hazelton D, Qian X. Endogenous regulation of angiogenesis in the rat aorta model. Role of vascular endothelial growth factor.  Am J Pathol. 1997;  151 1379-86.
• 26 Greenhalgh D G. The role of growth factors in wound healing.  J Trauma. 1996;  41 159-67.
• 27 Nicosia R F, Nicosia S V, Smith M. Vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro .  Am J Pathol. 1994;  145 1023-9.
• 28 de Vries C, Escobedo J A, Ueno H, Houck K, Ferrara N, Williams L T. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.  Science. 1992;  255 989-91.
• 29 Pajusola K, Aprelikova O, Pelicci G, Weich H, Claesson-Welsh L, Alitalo K. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.  Oncogene. 1994;  9 3545-55.
• 30 Terman B I, Dougher-Vermazen M, Carrion M E, Dimitrov D, Armellino D C, Gospodarowicz D. et al . Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.  Biochem Biophys Res Commun. 1992;  187 1579-86.