Opioid Receptors Stimulate Angiogenesis, Lymphangiogenesis and Neurogenesis in Ischemic Wounds in Sickle Mice.
Authors: Gupta Kalpna , Li Yunfang , Mason Nicholas A. , Vang Tou S. , Levay-Young Brett K. , Erickson Marna E. , Hebbel Robert P. 
Published: April 2020

Abstract
Painful leg ulcers are a serious manifestation of sickle cell disease. We showed that topically applied opioids stimulate angiogenesis and promote wound healing in rats, and mice expressing sickle hemoglobin. We hypothesized that opioids orchestrate the normal healing process by stimulating angiogenesis, lymphangiogenesis and neurogenesis. Therefore, we examined mechanism(s) associated with opioid receptor-mediated wound repair using (a) ischemic wounds on transgenic sickle mice with medium severity (hBERK) and control mice expressing normal human hemoglobin (HbABERK), and (b) an in vitro model of skin repair using human epidermal keratinocytes (HEK) and dermal microvascular endothelial cells (HDMEC). We observed HEK secreted β-endorphin into the culture medium ‘immediately’ after injury (9.6±1.3 ng/ml and 2.87±0.37 ng/ml after 30 min of injury), declining to undetectable levels 60 min post-injury. Culture medium from ‘immediately’ injured HEK stimulated about 30% increase in HDMEC proliferation (p &lt; 0.03 vs medium from intact HEK or medium from HEK 24h post-injury), which was completely antagonized by naloxone (1 μM), suggesting an opioid receptor-mediated effect. Furthermore, wounded monolayers of HDMEC incubated with ‘immediately’ injured HEK medium completely healed after 3 d as compared to 4 d with uninjured HEK medium. These data indicate endorphins secreted from skin keratinocytes upon injury stimulate endothelial proliferation, migration and angiogenesis via opioid receptors on endothelium. Therefore, we then examined wound scars from hBERK and HbABERK treated topically with either PBS/cream or morphine (3 mg/g cream) for 13 d. Z-series images were acquired using laser scanning confocal microscopy after immunostaining 100 micron thick wound cryosections for blood vessels, nerves and lymphatics, with anti-CD31, anti-PGP9.5 and anti-lymphatic vessel endothelium marker, respectively. PBS-treated hBERK wounds showed disorganized and stringy blood vessels, nerves and lymphatics, confined to the epidermis vs morphine-treated wounds showing normal architecture and their dermal as well as sub-epidermal localization, similar to that observed in HbA BERK wounds. Stereological quantitation revealed a significantly higher number of blood vessels and nerves in morphine vs PBS treated hBERK wounds (p&lt;0.05). Blood flow estimated by measuring 86Rb uptake by wound scars after tail vein injection showed a 2-fold increase in blood flow in morphine vs PBS treated hBERK wounds (p&lt;0.03), suggesting that opioids stimulate functionally normal vessels in the wounds. Opioid receptors (mu, delta and kappa) co-localized with blood vessels in both HbA and hBERK wounds but, the protein expression of only mu opioid receptor (MOR) was appreciably upregulated by morphine treatment of hBERK wounds as early as 3 d and also after 13 d as observed by Western immunoblotting. In HbABERK morphine-induced MOR upregulation occurred on 3 d but not 13 d. Wounds completely healed 13 d after morphine treatment in HbABERK but not in hBERK. Thus, MOR may be downregulated once the healing has occurred. These data suggest opioids, via their opioid receptors, stimulate endothelial proliferation and normal angiogenesis, lymphangiogenesis and neurogenesis. We speculate that in sickle cell disease where vasculopathy underlies the pathogenesis of painful leg ulcers, topically applied opioids may accelerate wound healing and may even provide pain relief.