Thrombospondin-1-CD47 blockade and exogenous nitrite enhance ischemic tissue survival, blood flow and angiogenesis via coupled NO-cGMP pathway activation

Abstract

Tissue ischemia and ischemia-reperfusion (I/R) remain sources of cell and tissue death. Inability to restore blood flow and limit reperfusion injury represents a challenge in surgical tissue repair and transplantation. Nitric oxide (NO) is a central regulator of blood flow, reperfusion signaling and angiogenesis. De novo NO synthesis requires oxygen and is limited in ischemic vascular territories. Nitrite (NO(2-)) has been discovered to convert to NO via heme-based reduction during hypoxia, providing a NO synthase independent and oxygen-independent NO source. Furthermore, blockade of the matrix protein thrombospondin-1 (TSP1) or its receptor CD47 has been shown to promote downstream NO signaling via soluble guanylate cyclase (sGC) and cGMP-dependant kinase. We hypothesized that nitrite would provide an ischemic NO source that could be potentiated by TSP1-CD47 blockade enhancing ischemic tissue survival, blood flow and angiogenesis. Both low dose nitrite and direct blockade of TSP1-CD47 interaction using antibodies or gene silencing increased acute blood flow and late tissue survival in ischemic full thickness flaps. Nitrite and TSP1 blockade both enhanced in vitro and in vivo angiogenic responses. The nitrite effect could be abolished by inhibition of sGC and cGMP signaling. Potential therapeutic synergy was tested in a more severe ischemic flap model. We found that combined therapy with nitrite and TSP1-CD47 blockade enhanced flap perfusion, survival and angiogenesis to a greater extent than either agent alone, providing approximately 100% flap survival. These data provide a new therapeutic paradigm for hypoxic NO signaling through enhanced cGMP mediated by TSP1-CD47 blockade and nitrite delivery.

Figure 1. Nitrite increases ischemic soft tissue survival

(A) Random dorsal myocutaneous flaps in C57BL/6 mice were created. Animals were randomized into three groups, untreated, vehicle alone and nitrite (48 nmols/animal i.p.). Flap survival was evaluated 7 days after surgery. (B) Acute changes in blood flow were determined via laser Doppler during the first post-operative hour. * = p<0.05 compared to vehicle. (C) Nitric oxide levels were determined via chemiluminescence from acutely excised ischemic soft tissue flaps following nitrite treatment (10 mM) in the presence or absence of allopurinol (500 µ) or potassium hexacyanoferrate (500 µM). (C) Representative raw data and (D) quantification of NO generation under the indicated treatment conditions. Results presented are the mean ± SD, n = 3 in each treatment group. * = p<0.05.

Figure 2. Nitrite enhancement of ischemic flap survival is abrogated by inhibition of sGC activation

(A) Random dorsal myocutaneous flaps 1 × 3 cm in dimensions were created in male C57BL/6 mice. Animals received nitrite (48 nmols/animal) or nitrite and ODQ (20 µg/g i.p.). Flap survival was analyzed on post-operative day 7 Results presented are the mean ± SD of 5 animals in each group. * = p<0.05 compared to nitrite alone. (B) Flap blood flow was assessed during the immediate post-operative interval via laser Doppler. Results presented are the mean ± SD of 5 animals in each group. * = p<0.05 compared to nitrite plus ODQ.

Figure 3. Myocutaneous tissue survival to ischemia is increased by combined TSP1 targeting and nitrite

(A) Random dorsal myocutaneous flaps 1 × 3 cm in dimension were created in male C57BL6 mice and evaluated on post-operative day 7. Animals received an IgG2 control Ab, a TSP1 monoclonal antibody (clone Ab A6.1) (both at 2.4 µg per tissue unit), nitrite (48 nmols/animal) plus an IgG2 Ab, or nitrite plus the A6.1. Representative flaps were photographed. (B) Flap survival is expressed as percent of total and was determined as described in the methods. Results are the mean ± SD of 5 animals in each group. * = p<0.05 compared to isotype control Ab or Ab A6.1 alone. (C) A dose range of TSP1 antibody A6.1 (0.3, 0.6, 1.2 and 2.4 µg per tissue unit) was tested against an optimum amount of nitrite (48 nmols/animal) and flap survival determined. * = p<0.05 compared to nitrite alone. (D) Tissue cGMP was determined by ELISA from flaps on post-operative day 7 and results normalized to total protein. Results are the mean ± SD of 5 animals in each group. * = p<0.05 compared to Ab A6.1 alone.

Figure 4. Concurrent CD47 suppression and nitrite supplementation increases ischemic tissue survival beyond single agent therapy

(A) Random dorsal myocutaneous flaps 1 × 3 cm in dimension were created in sex and age matched C57BL/6 mice. Animals received a CD47 suppressing morpholino or a mismatch control morpholino alone or in combination with nitrite at the time of surgery. (B) Flap survival was assessed on post-operative day seven. Results represent the mean ± SD of 5 animals in each group. * = p<0.05 compared to CD47 morpholino alone.

Figure 5. Nitrite and TSP1-CD47 blocking antibodies increase muscle explant angiogenic response

(A) Pectoralis major muscle biopsies (1 mm₃) excised from C57BL/6 mice were embedded in 3D type I collagen matrix and incubated in growth medium ± nitrite (0.1 – 10 µM) ± a TSP1 monoclonal antibody (clone Ab 6.1 (1 µg/ml)) or ± a CD47 monoclonal antibody (clone 301 (1 µg/ml)). (B) Angiogenic response was quantified 4 days post-explantation Photomicrographs were obtained at 10x. Results represent the mean ± SD of three separate experiments.

Figure 6. Nitrite and TSP1-CD47 blockade enhances macro-vascularity of random ischemic soft tissue flaps

(A) Random dorsal myocutaneous flaps 1 × 3 cm in dimensions were created in male C57BL/6 mice. Animals received the indicated treatments at reported dosages (see Fig 2). On the 7_th post-operative day animals were anesthetized and the flap undersurface digitally photographed. (B) Images were later quantified as described in the methods section to yield a vascularity index. Results presented are the mean ± SD of 5 animals in each treatment group. * = p<0.05 compared to a matched isotype control Ab.

Figure 7. Nitrite and TSP1-CD47 blockade enhance micro-vascularity of random ischemic soft tissue flaps

(A) Random dorsal myocutaneous flaps were excised, fixed and stained with hematoxylin and eosin. (B) Flaps were then visualized under 10x magnification and patent and blood filled vessels were counted. Results are from 5 flaps in each treatment category. * = p<0.05 compared to a matched isotype control Ab. Blue arrows indicated vessels containing red blood cells.

Figure 8. Nitrite and TSP1-CD47 signaling pathways converge at sGC

Tissue ischemia arises from a decreased blood flow. As blood flow decreases tissue perfusion and oxygen drop and pH increases. In this microenvironment nitrite acts as an NO prodrug being converted by endogenous reductase activity to NO. Thus exogenous nitrite selectively delivers exogenous NO to areas where it is most needed. NO activates sGC to increase cGMP synthesis. cGMP acts on several downstream targets in endothelial cells to stimulate pro-angiogenic responses and in vascular smooth muscle cells to dilate blood vessels. At physiological concentrations, TSP1 acts primarily through its receptor CD47 to limit sGC activation [16]. At nM concentrations, TSP1 also signals through CD36 to inhibit the same responses [51], but CD47 is also necessary for these signals to inhibit cGMP signaling. Blockade of TSP1-CD47 signaling with monoclonal antibodies to TSP1 (Fig. 3) and CD47 or gene silencing of CD47 (Fig. 4) increases the pro-survival activities of NO and enhances angiogenesis, tissue perfusion and blood flow and survival from ischemia.