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Effects of Synthetic Peptides on Angiogenesis and Wound Healing

CM Crandell, Y E, HE Baldwin, WL Lee, and AR SHalita
Department of Dermatology, State University of New york Downstate Medical center,
Brooklyn, NY.


Introduction
The incidence of chronic non-healing wounds in the United States is vast. It is estimated that more than five million people suffer annually from this condition. The resultant human and health care costs are immeasurable. Current therapies include topical, systemic, and absorptive modalities that can be costly, cumbersome, and/or time-consuming. Treatment is at times unsatisfactory, with approximately 60% of such lesions recurring. Angiogenesis, the growth of new blood vessels, represents a critical step in the wound healing cascade. It is a complex cellular process, of which cell migration is a critical component. Numerous peptides exist which are known to be important mediators that facilitate Angiogenesis. ACT 1 is a novel, 28 amino acid linear polypeptide that may be derived by cleavage of thymosin. ACT2 is a similar 44 amino acid linear peptide. Both synthetic peptides have been shown to mediate endothelial cell migration and proliferation in vitro. Moreover, in vivo assays performed with ACT 1 have resulted in augmented wound healing. This, we propose that when applied topically, these synthesized peptides will expedite the wound healing process, especially in recipients with impaired capacity for Angiogenesis.


Objectives
To determine the in vitro effects of ACT 1 and ACT 2 on endothelial cell migration and Angiogenesis, in comparison with known growth factors.
To determine the effects of intraperitoneal ACT 1 using a rat wound healing model.
To ascertain the ability of topical ACT 1 to speed wound healing in a human subject with impaired angiogenic potential.


Results


Figure 1: This graph compares the effects of three positive controls, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (FGF), on endothelial cell migration as measured by a Boyden Chamber Chemotactic Assay (1). When growth factors are employed as chemoattractants for endothelial cells, it is evident that both VEGF and PDGF possess a greater ability to attract endothelial cells than FGF. Based on these results, we chose VEGF as our positive control for the remaining in Vitro assays.







Figure 2: The comparative effects of variable concentrations of ACT1 and ACT2 on endothelial cell migration as measured by Boyden Chamber assays are depicted above. These data were obtained by comparing peptide treated versus positive control (VEGF) treated endothelial cell migration. It appears that both peptides act as chemattractants for endothelial cells, comparable to VEGF. In addition, a dose response is evident, with both ACT1 and ACT2 exerting maximal affects on cell migration at a concentration of 100ng/ml.











Figure 3: This graph reveals the comparative effects of constant doses of ACT 1 and ACT 2 (10ng/ml) on endothelial cell migration versus our positive controls as measures by a scratch-wound closure assay (1). Here confluent monolayers of endothelial cells were scratch “wounded” with the tip of a universal blue pipatte and incubated with ACT 1 and ACT 2, along with two positive controls, VEGF and FGF and incubated with (also at 10ng/ml), for 4,8 and 24 hours. Migration of cells into the wounded area after 24 hours was increased in the presence of ACT 1 and ACT 2, 4-fold and 3-fold, respectively, when compared to migration with media alone (bottom gray line)


ACT1 - Gap Closure and Wound Width in IP Findings:
For the in vivo effects of intraperitoneal ACT 1 as measured by a rat wound healing model, full-thickness 8mm punch biopsy wounds were made on the dorsal surface of rats (2). ACT 1 (60 micrograms in 300 microliters normal saline) was injected intraperitoneally on injury date and every other day thereafter. Likewise, identical amounts of normal saline (NS) were injected intraperitoneally to control animals. Intraperitoneal injection of ACT 1 between days 4-7 resulted in a 42% (+/- 9%) and an 18% decrease in gap length and wound width, respectively, when compared to NS alone.


Conclusions

  • ACT 1 and ACT 2 facilitate endothelial cell migration as evidenced by the Boyden Chamber and scratch-wound closure assays.

  • When compared to VEGF, PDGF, and FGF, both ACT 1 and ACT 2 possess a similar potential to enhance Angiogenesis.

  • Intrapertoneal ACT 1 appears to significantly decrease the gap size and wound width of artificial wounds in a rat wound-healing model system. These findings lend support to the high diffusibility of ACT 1 in tissues.

  • Topical ACT 1 appears to enhance the wound-healing capacity of subjects with impaired angiogenic potential. We believe that when administered topically to resistant wounds, ACT 1 speeds the wound-healing cascade by facilitating Angiogenesis. Clinical studies comparing the efficacy of ACT 1 to those of commercially available products such as genetically-engineered PDGF (indicated for use in diabetic ulcers) are in progress to further elucidate the role of ACT 1 in the chronic wound milieu.


Reference:

  1. Malinda, K.M. Goldstein, A.L. and Kleinman, H.K. (1997). Thymosin Beta 4 Stimulates directional migration of human umbilical vein endothelial cells, FASEB J. 11:474

  2. Malinda, K.M. Sidhu, G.S., Banaudha, K.K. Gaddipati, J.P. Maheshwari, R.K., Goldstein A.L., Kleinman, H.K. (1998). Thymosin Alpha 1 stimulates endothelial cell migration, Angiogenesis, and wound healing. J. Immunology, 160:1001-1006

  3. Rothe M. Falanga, V (1989). Growth Factors: Their biology and promise in dermatologic diseases and tissue repair. Arch. Dermatology 125: 1390-8

  4. Valencia, I.C. Falabella, A., Kirsner, R.S., and Eaglestein, W.H. (2001) Chronic venous insufficiency and venous leg ulceration. J.Amer. Acad. Dermatology. 44, 3:401-402.