Algae-Derived Omega-7 Accelerates Wound Healing

1. Sheep: 672M Control Omega-7 Algae-Derived Omega-7 Accelerates Wound Healing R. Connelly, M. Montoya, D. Schmid, R. Pearsall, M. Werst, R. Hebner University of Texas at Austin, Center for Electromechanics and OpenAlgae LLC Time Control Phases Main Cell Types Omega-7 Specific Events 42L media Healthy Wound Diabetic Wound Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia Platelet aggregation, release of fibrinogen and other pro-inflammatory mediators Migrating keratinocytes Platelets 7 days post surgery 4/5 of stock 24L media 48L algae Hours Neutrophils, monocytes Post Op 7 days Selectinsslow down neutrophils, diapedesis through integrin binding 12L media VEGF VEGF 24L algae Inflammation cell recruitment and chemotaxis, wound debridement 12L stock Control mouse Diabetic mouse 15L bioreactor keratinocytes Matrigel 3D culture 95L bioreactor Macrophages collagen deposition fibroblasts Day 1 Day 5 Day 8 Stock culture Day 3 Hemidesmosomebreakdown, keratinocyte migration MF Days MP Neovasculogenesis and wound healing Impaired neovasculogenesis and wound healing Concentrate 100X Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction Keratinocytes, fibroblasts, endothelial cells Large animal studies In vivo mouse studies 1 In vitro 3D cell culture 14 days post surgery 2 3 Methods Significance of the Study Preliminary Results Crosstalk between MMPs, VEGF signaling, NOS activation, EPC production and migration, ECM production EPCs homing to wound site Limited EPC homing to wound site Myofibroblasts • Diabetes affects approximately 170 million people worldwide, including 20.8 million in the USA, and by 2030 these numbers are projected to double. • Impaired wound healing is a major clinical problem in diabetic patients, affecting about 15 percent of them and is the leading cause of lower limb amputations. • Wound healing progresses in phases: keratinocytes Post Op 18 days Weeks to months Matrigel 3D culture Remodeling scar formation and revision, ECM degradation, further contraction Phenotypic switch to myofibroblasts from fibroblasts • Treat with omega-7 prior to wound formation, then analyze: • Collagen deposition (western blot, microscopy) • VEGF activation (western blot, microscopy) • NOS activation (ROS formation DCF fluorescence) • Cellular proliferation (XTT assay) • Wound closure (SOC measurements) • Scar formation (SOC measurements) Circulatory system fibroblasts 18 days post surgery eNOS activation Endothelial Progenitor Cell (EPC) mobilization into the circulation Impaired eNOS activation Limited NO. Limited EPC mobilization into the circulation ↑ NO. Recover Omega-7 Oil Exposure to an Electric Field Bone marrow MP – macrophage MF – myofibroblast Figure 4. Ultrasound assessment of skin graft healing 7 and 18 days post surgery.Untreated control wound between the mesh was epithelialized slowly and irregularly. The Omega-7 treated wound epithelialized rapidly and smoothly. Preliminary Results Blood flow measured in donor and grafted side by dopler Fatty Acid Algae Oil Extract 350 – Graft Treatment 300 – 16:0 16:1 (n-7) 18:1 (n-9) 18:2 (n-6) 18:3 (n-3) other 30.9 33.5 14.6 5.3 5.7 10.0 Conclusions • One of the cardinal features of wound healing is the formation of new small blood vessels at the site of injury. 250 – Graft Control 200 – • We have identified an omega-7-rich algae that is appropriate for large scale production. • This algae can be effectively opened by exposure to an appropriate electric field. • Omega-7 can be recovered using column chromatography. • Omega-7 extracts improve blood flow in wounded sheep. • Omega-7 accelerates wound closure and minimizes scar formation. Donor Treatment 150 – 100 – Donor Control Figure 1. Fatty Acid composition of the algal extract. The primary fatty acids present in this algae are palmitic and palmitoleic (Omega-7) fatty acids. 50 – 0 – 7 d 18 d Figure 2. Blood flow in control and omega-7 treated sheep. Sheep were given 10 mm surface wounds, then were treated with omega-7 or nothing. Blood flow is increased in omega-7 treated sheep, suggesting increased neovasculargenesis at the wound site. Figure 3. Wound closure.The untreated control wound failed to fully close by 18 days post surgery and scar tissue was apparent. The Omega-7 treated wound closed 97% by day 14, and completely by day 18 post surgery. The epithelium was smooth with minor scarring. • The essential fatty acid palmitoleic acid (Omega-7) promotes the formation of new blood vessels and collagen deposition at the site of injury. • Currently, Omega-7 is primarily derived from Sea Buckthorn, a cold weather plant that is harvested once a year. • We have identified an algae that produces large quantities of Omega-7 and can be harvested daily.

2. Healthy Wound Diabetic Wound migrating keratinocytes VEGF VEGF collagen deposition MF MP neovasculogenesis and wound healing impaired neovasculogenesis and wound healing EPCs homing to wound site limited EPC homing to wound site circulatory system eNOS activation Endothelial Progenitor Cell (EPC) mobilization into the circulation impaired eNOS activation limited NO. limited EPC mobilization into the circulation ↑ NO. bone marrow MP – macrophage MF – myofibroblast

3. Time Phases Main Cell Types Specific Events Coagulation fibrin plug formation, release of growth factors, cytokines, hypoxia platelet aggregation, release of fibrinogen and other pro-inflammatory mediators platelets hours neutrophils, monocytes selectins slow down neutrophils, diapedesis through integrin binding Inflammation cell recruitment and chemotaxis, wound debridement macrophages hemidesmosome breakdown, keratinocyte migration days keratinocytes, fibroblasts, endothelial cells Migration/ proliferation collagen deposition, angiogenesis, ECM deposition, contraction crosstalk between MMPs, VEGF signaling, NOS activation, EPC production and migration, ECM production myofibroblasts weeks to months Remodeling scar formation and revision, ECM degradation, further contraction phenotypic switch to myofibroblasts from fibroblasts

4. 42L media 4/5 of stock 24L media 48L algae 12L media 24L algae 12L stock 15L bioreactor 95L bioreactor stock culture Day 1 Day 3 Day 5 Day 8 concentrate 100X Recover Omega-7 Oil Exposure to an Electric Field

5. diabetic mouse control mouse 1 in vitro 3D cell culture in vivo mouse studies large animal studies 2 3 keratinocytes Matrigel 3D culture fibroblasts • Treat with omega-7 prior to wound formation, then analyze: • collagen deposition (western blot, microscopy) • VEGF activation (western blot, microscopy) • NOS activation (ROS formation DCF fluorescence) • cellular proliferation (XTT assay) • wound closure (SOC measurements) • scar formation (SOC measurements)

6. Blood flow measured in donor and grafted side by dopler • Blood flow is greater in treated wound with omega-7 oil

7. control Omega-7 Donor side 7 days post surgery 14 days post surgery 18 days post surgery no remarkable difference -epithelization of treated wound was faster treated wound: almost 100％ untreated wound: 78％ -no remarkable difference -treated wound ; almost 100％ -untreated wound ; 97％

8. control Omega-7 Post Op 7 days Post Op 18 days Sheep: 672M The wound between the mesh in untreated part was not epithelized. Treated wound epithelized partially. Untreated wound between mesh was epthelized irregularly. Treated wound between mesh was epithelizedequally.