Intracellular ATP Delivery Causes Rapid Tissue Regeneration via Upregulation of Cytokines, Chemokines, and Stem Cells

Abstract

We have reported accelerated wound healing induced by intracellular ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of cytokines, growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four wounds were created on the ventral side of each ear. Two wounds on one side were treated with ATP-vesicles (10 mM ATP), and the other two were treated with controls (normal saline or Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for cytokines and growth factors using real-time PCR and immunohistochemical staining. The control wounds showed an immediate increase in proinflammatory cytokines after wound creation but no further increase after this initial spike. The growth factor levels in the control wounds remained unchanged throughout the study. Conversely, the wounds treated with ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1b and TNF-a expression from day 1-4, and significantly higher VEGF-A, VEGF-D, and VEGFR-2 expression from day 4-6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage collagen production and neovascularization.

Enhanced Skin Incisional Wound Healing With Intracellular ATP Delivery via Macrophage Proliferation and Direct Collagen Production

“…Scale bar is 50 μm. function of the wound cells during the early ischemic stage (Sarojini et al, 2017;Mo et al, 2020). The healing events occurred in the current study also shows the same progression.…”

“…Our excisional wound study revealed a unique phenomenon when ATP-vesicles were used: very early and massive macrophage accumulation, in situ proliferation, and M2 polarization ( Wang et al, 2009a ; Wang et al, 2010 ; Howard et al, 2014 ; Sarojini et al, 2017 ; Mo et al, 2020 ). The M2 polarized macrophages produce collagen directly to enhance healing at early time.…”

“…In previous studies we reported positive effect of intracellular delivery of Mg-ATP (ATP-vesicles) to enhance excisional skin wound healing process in non-ischemic and ischemic wounds both in non-diabetic and diabetic animals ( Chien, 2006 ; Wang et al, 2009a ; Wang et al, 2010 ; Chien, 2010a ; Chien, 2010b ; Howard et al, 2014 ; Sarojini et al, 2017 ; Mo et al, 2020 ). One unique feature of intracellular delivery of ATP-vesicles was the very early and massive macrophage accumulation, and these studies provide the primary evidence of in situ macrophage proliferation, M2 polarization, and direct collagen production during wound healing ( Chien, 2006 ; Wang et al, 2010 ; Chien, 2010a ; Chien, 2010b ; Sarojini et al, 2017 ; Mo et al, 2020 ) without the involvement of other known factors such as parasite infection ( Jenkins et al, 2011 ). This feature could be especially valuable for the healing of incisional wounds because the healing of incisional wounds depends more heavily on the formation of the extracellular matrix, especially tissue collagen content ( Klinge et al, 2006 ; Chang et al, 2010 ; Petersson et al, 2014 ).…”

Innate Immunity in Diabetic Wound Healing: Focus on the Mastermind Hidden in Chronic Inflammatory

“…Moreover, innate immune cells such as neutrophils and macrophages continuously infiltrate during the inflammatory phase and interact with the high expression of chemokines, Macrophage inflammatory protein-2 (MIP-2) and Monocyte chemotactic protein-1 (MCP-1), up-regulate inflammatory mediators IL-1β, TNF-α, etc. result in continual amplification of signals of inflammatory until 13 days or more after injury (Wetzler et al, 2000;Tousoulis et al, 2013;Mo et al, 2019). Meanwhile, the continual differentiation and formation of proinflammatory macrophages can also stimulate the synthesis of Matrix metalloproteinases (MMPs) jointly with TNF-α, cause excessive destruction of extracellular Matrix and damaged granulation tissue formation (Acosta et al, 2008), inhibit proliferation and migration of fibroblasts and angiogenesis (Xu et al, 2013), activate innate immunity through ROS generated by oxidative stress (Warnatsch et al, 2015), activate NLRP3 inflammasome and lead to exacerbating wound inflammation (Lee et al, 2013;Mirza et al, 2014;Dai et al, 2017).…”

“…Under the pathological conditions of stress and inflammatory, caspase-1 released after the activation of NLRP3 inflammasome can also mediate a rapid programmed cell death pattern characterized by accompanying inflammatory response, called "Pyroptosis" (Wu et al, 2018). Pyroptosis induces the release of a large number of pro-inflammatory factors, eventually forming a cascade of amplified inflammatory responses (Jorgensen and Miao, 2015;Wang S. et al, 2019). In the state of DW, ROS and IL-1β can activate the NLRP3 inflammasome in Mp, promote the maturation and secretion of IL-1β, aggravate local inflammation through positive feedback, and hinder wound healing (Lee et al, 2013;Mirza et al, 2014;Dai et al, 2017).…”