Abnormal Angiogenesis, Neurogenesis and Lymphangiogenesis in the Skin Underlies Delayed Wound Healing in the Sickle Mouse: Acceleration of Healing by Topical Opioids.
Authors: Gupta Kalpna , Poonawala Tasneem , Levay-Young Brett K. , Ericson Marna E. , Ammbashankar Niroop S. , Hebbel Robert P. 
Published: April 2020

Abstract
Chronic non-healing and painful leg ulcers are a serious manifestation of sickle cell disease (SCD). We hypothesized that structurally and functionally aberrant interdependent triad of angiogenesis, neurogenesis and lymphangiogenesis leads to localized ischemic insult, pain and edema, respectively, resulting in non-healing, painful and ulcerative wounds in SCD. We examined the dorsal skin of sickle mice with different severity (mild, NY1DD, medium, hBERK and severe, BERK) for blood vessels, sensory nerves and lymphatic vessels, by immunostaining 100 micron thick cryosections with endothelial specific anti-CD31, PGP9.5 and calcitonin gene related peptide (CGRP) and anti-lymphatic vessel endothelium marker LYVE-1, respectively. Z-series images were acquired using using laser scanning confocal microscopy. We observed a drastic difference in the structure, presentation and localization of all three processes in sickle mice as compared to their controls, which increased with the severity of the model. We found that, [i] epidermal thickening in BERK is 1/2 vs HbABERK (control expressing normal human Hb), [ii] majority of blood vessels localized in the sub-epidermal region in BERK vs dermal tissue in controls, [iii] microvasculature shows disorganization,tortuous, stringy and collapsed vessels in sickle, [iv] open lymphatic vessels arranged in parallel union with hair follicles in controls collapsed to 50 and 90% in diameter in mild and severe sickle models, respectively, while they moved by 90° becoming perpendicular to hair follicles in the severe sickle; [v] Peptidergic-immunoreactive nerve fibres increased with the severity of sickle state in the epidermis; and [vi] CGRP, a mediator in pain transmission is upregulated 2–5 times in sickle vs controls. These data suggest that aberrant, non-functional blood vessels lead to the development of localized hypoxia and ischemia resulting in tissue damage and compression of lymphatics which in turn increase edema and ulceration. Upregulated CGRP on peripheral nerves is suggestive of severe pain associated with sickle ulcers. Opioids interact with endothelium as well as nerves and promote angiogenesis and analgesia, respectively. Therefore, we examined the effect of topically applied opioids on healing of ischemic wounds in hBERK. In PBS treated HbABERK control, wound healing was 62% vs 42% in sickle on day 12 (p&lt; 0.02). Complete wound closure was observed on day 19 in HbABERK, whereas, complete healing did not occur in sickle mice until 28 days. Topical application of morphine resulted in complete closure of wounds by days 14 and 23 in HbABERK and sickle mice, respectively. Similarly, hydromorphone stimulated complete wound closure as compared to 85% closure with PBS by day 23 in sickle (p&lt;0.03). Blood and lymphatic vessel lengths were 20–35% higher in morphine or hydromorphone vs PBS-cream treated sickle wounds on days 13 and 28, respectively (p&lt;0.05 PBS-Cream vs others on days 13 and 28). Thus wound repair is delayed in SCD due to aberrant vasculature and lymphatics and opioids stimulate normal angiogenesis and lymphangiogenesis which promote wound healing. We speculate that topical opioids may accelerate wound healing in painful non-healing leg ulcers in SCD.