Abstract
Skin flap ischemia-reperfusion (IR) injury is the key factor to the success rate of skin transplantation, the molecular mechanism of flap IR injury needs to be continuously explored to provide new ideas for its clinical treatment. G protein-coupled receptor kinase 2 (GRK2) was reported to be involved in regulating mitochondrial function, and mitochondria were essential in the process of flap IR. Thus, we aimed to investigate the function of GRK2 in flap ischemia-reperfusion injury and further explore the underlying mechanism. Sixty male C57BL/6 mice were randomly divided into four groups: sham, IR+sh-NC, IR+sh-GRK2 and IR+sh-GRK2+ dynamin-related GTPase 1 (Drp1). Flap function and mitochondrial function were determined after ischemia for 3 hours and reperfusion for 72 hours. Comparing with sham group, GRK2 was increased in flap after IR injury. Loss of GRK2 inhibited cell apoptosis and promoted cell proliferation of flap after IR injury. And deficiency of GRK2 promoted mitochondrial function in flap after IR injury. IR injury up-regulated Drp1 expression in flap, while sh-GRK2 down-regulated Drp1 expression. Furthermore, overexpression of Drp1 removed the protective effect of sh-GRK2. In conclusion, our study revealed that GRK2 deletion improved flap function and mitochondrial function by inhibiting Drp1 expression, which may provide a new insight for the clinical treatment of flap ischemia-reperfusion injury.
Keywords: Skin flap ischemia-reperfusion injury, ROS, GRK2, Drp1, mitochondrial function
Introduction
Skin transplantation is the main method to repair the defects and malformations of body surface tissues and organs caused by tumor resection and various trauma [1]. Therefore, the survival of transplanted tissue often determines the success or failure of the operation. After a period of flap ischemia, the restoration of blood flow in the flap not only fails to restore its function, but aggravates the structural damage and dysfunction, and results in ischemia-reperfusion (IR) injury [2]. After years of research, the survival rate of the flap has been improved after transplantation, but there are still some cases of partial or total tissue necrosis in the clinic [3]. Therefore, the molecular mechanism of skin flap IR injury needs to be continuously explored to provide new ideas for its clinical treatment.
A large number of reactive oxygen species (ROS) will be produced in the process of IR of the skin flap, and hypoxanthine will accumulate in the body during ischemia-ischemia [4]. During the process of restoring blood flow, xanthine oxidase will convert hypoxanthine into guanosine and superoxide ion [5]. Studies have shown that [6] IR injury of skin flap positively regulates xanthine oxidase, which can improve ROS level in tissues, and the positive regulation of xanthine oxidase also mediates the inflammatory response in tissues [7]. During IR, neutrophils are activated and release a variety of cytokines, which in turn activate NADPH, causing an oxygen burst and releasing a large number of oxygen free radicals [8]. Thus, mitochondria, the main place for producing ROS, were important in the process of skin flap IR [9].
G protein-coupled receptor kinase (GRKs) is a critical serine/threonine kinases in the body, which can phosphorylate G protein-coupled receptors (GPCR), and thus participate in the regulation of GPCR signal and the regulation of multiple ligands and drug effects [10]. In recent years, some studies have found that GRK2 is involved in regulating mitochondrial function [11,12]. During myocardial ischemia-reperfusion injury, GRK2 is transferred from cytoplasm to mitochondria, thereby promoting oxidative stress response and initiating pro-apoptosis procedures [12]. Meanwhile, GRK2 can promote the accumulation of superoxide and damage mitochondrial function [13]. Currently, little is known about the function of GRK2 in skin flap ischemia-reperfusion injury. Thus, we aimed to investigate the function of GRK2 in skin flap ischemia-reperfusion injury and further explore the underlying mechanism.
Materials and methods
Animal experiment and grouping
Sixty male C57BL/6 mice were randomly divided into four groups: sham, IR+sh-NC, IR+sh-GRK2 and IR+sh-GRK2+Drp1, n = 15 for each group. The mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (80 mg/kg), and then fixed to the operating board after they lost consciousness. The abdominal hair was shaved with a razor blade, and the midline of the mice abdomen was used as the axis of symmetry. The protruding part of the sternum from the upper to the groin was designed and labeled as a 4 cm×1 cm island flap. The operation and the surrounding area were sterilized with iodine, and the flap was gradually stripped down along the marked line to separate it from the muscle layer. When the left and right inferior superficial arteries were peeled, the connection between the vessels on both sides and the flap should be ensured. To ensure that only one side of the superficial artery supplied blood to the flap, the inferior superficial artery of the right abdominal wall was cut off by ligation, and the inferior superficial artery of the left abdominal wall was closed by vascular clamp for 3 hours. After the preparation of the flap was completed, to prevent the implantation of new capillaries in the muscle layer, a 0.1 mm thick silicone membrane was inserted between the flap and the abdominal muscle layer, and then the flap was sutured. The sham group was not subjected to ischemia, but ligate the left superficial epigastric artery. Adeno-associated virus 9 (AAV9) vector containing GRK2-shRNA (AAV9-sh-GRK2) or AAV9-Drp1 (6×1011 GC) was injected into mice through tail vein for 4 weeks, then mice were suffered from IR surgery. The experiments were finished depended on the protocols and accordance to the National Institutes of Health guidelines. This study was reviewed and approved by the Institutional animal care and use committee of Wuxi No. 9 People’s Hospital Affiliated to Soochow University.
TUNLE staining
We used the in situ Cell Death Detection Kit (TUNEL fluorescence FITC kit, Roche, Germany) detect apoptotic level of skin flap. DAPI was used to stain nuclei. IX73 fluorescence microscope (Olympus, Valley, PA) was used to analyze fluorescence staining. The total cells and TUNEL positive cells numbers was counted by Image-J.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from serum and culture medium according to a standard protocol. And then, the purity and concentration of RNA was detected and all the samples were converted into cDNA using reverse transcription kit. We used SYBR Green (Thermo Fisher Scientific) system to perform the qRT-PCR. Primers list: GRK2: Forward primer: 5’-CTTCCAGCCATACATTGAGGAG-3’, Reverse primer: 5’-TTCGATGCACACTGAAGTCATT-3’, Drp1: Forward primer: 5’-TCCCTAAACTCCATGATGCCATA-3’, Reverse primer: 5’-CCACAGGCATCAGCAAAGTC-3’, GAPDH: Forward primer: 5’-AGGTCGGAGTCAACGGATTTG-3’, Reverse primer: 5’-CCTGGAAGATGGTGATGGGAT-3’.
Western blot analysis
Protein samples were blotted depended on standard protocol. And we used Odyssey Infrared Scanning System (Gene Co. Ltd., Hongkong, China) to scan the membranes. At last, we used Image J software to analyze the western bolt results. The primary antibodies are as list: GRK2 (SAB4500592, Sigma-Aldrich), Drp1 (10656-1-AP, Proteintech) and GADPH (12935-1-AP, Proteintech). The secondary antibodies IRDye700/800 Mouse or Rabbit were produced by LICOR (Lincoln, Nebraska, USA).
Immunohistochemical (IHC) staining
Frozen sections of tumors were fixated in 4% paraformaldehyde and washed using PBS. We penetrated sections using 0.5% Triton X-100. After 3 times wash, we blocked sections with 50% goat serum. Then, sections were incubated with primary antibody overnight. Then, we incubated the sections using secondary antibody. Immunofluorescence was analyzed under an IX73 fluorescence microscope (Olympus, Valley, PA).
Hematoxylin and eosin (H&E) staining
The skin flap tissues were gathered and fixed in 4% paraformaldehyde for 24 h. Then the fixed tissues were embedded in paraffin. Next, Paraffin slicer machine was used to cut slices (5-mm cross-sectional). Skin flap sections were dewaxed with xylene and treated with ethanol at different concentrations for 5 minutes. Hematoxylin staining for 5 minutes, 5% acetic acid treatment for 1 minute, water rinse. Dye with eosin for 1 minute, rinse with running water. Dehydrate in 70%, 80%, 90%, 100% ethanol for 10 seconds, xylene for 1 minute. Drizzle with neutral gum and seal.
Mitochondrial assays
The mitochondria was isolated from skin (40-50 mg) as described [14]. ATP production was measured with ATP Bioluminescent Assay (Sigma). ATP content was determined by ATP Assay Kit (Beyotime, China). The final ATP content of each sample was normalized to its protein concentration with the BCA protein detection kit (Beyotime, China). Mitochondrial electron transport chain complex activities were calculated using MitoCheck Complex Activity Assay Kit (Cayman chemical, USA). Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience) was used to measure basal and maximum oxygen consumption rate (OCR) and proton leak.
Statistical analysis
All data is presented as a mean ± S.E.M. Statistical analysis was performed using Student’s t-test or Wilcoxon test or a one-way ANOVA.
Results
GRK2 was decreased in the skin after IR injury
To evaluate the expression of GRK2 in skin after IR injury, we first established IR model (ischemia for 3 hours and reperfusion for 72 hours) in the mice skin. As shown in Figure 1A, the skins in sham group were pink and elastic, while they were brown and inelastic in IR group, indicating a significant decrease of flap survival rate in IR group. As well, TUNEL analysis showed the number of apoptotic positive cells increased significantly in the IR group comparing with sham group (Figure 1B). Then, we performed a series of experiments to determine GRK2 expression in skin flap during IR injury. And we found that the mRNA and protein levels of GRK2 was upregulated in IR group (Figure 1C and 1D). Furthermore, IHC data showed IR injury induced GRK2 expression in skin flap (Figure 1E). Together, our results suggested that GRK2 was increased in skin flap after IR injury.
Figure 1.
The expression of GRK2 in skin flap after IR injury. IR model (ischemia for 3 hours and reperfusion for 72 hours) was established in the abdominal skin of mice. A. Representative photographs of abdominal skin flap, and the survival rate of the total flap area was calculated. B. TUNEL staining was used to determine apoptosis level. Bar = 100 μm. C. qRT-PCR assay analyzed the expression of GRK2 in skin flaps. D. Western blot detected the protein expression of GRK2 in skin flaps. E. IHC staining for GRK2 in skin flaps. Bar = 500 μm. n = 10, *P<0.05, **P<0.01 vs sham. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Knockdown of GRK2 enhanced skin function and cell proliferation after IR injury
To identify the function of GRK2 in the skin after IR injury, we injected adeno-associated virus 9 (AAV9) vector containing GRK2-shRNA (AAV9-sh-GRK2) into mice undergoing IR operation through tail vein. And the qRT-PCR analysis showed a significant decrease in the skin injected with AAV9-sh-GRK2 (Figure 2A). Then functional experiments showed that loss of GRK2 improved the color and elasticity, which showed an enhancive flap survival rate (Figure 2B). TUNEL staining showed sh-GRK2 reduced the number of apoptotic positive cells after IR operation (Figure 2C). H&E staining indicated that injection of sh-GRK2 suppressed the number of inflammatory cells after IR injury (Figure 2D). Then, inflammation cytokines were determined, and the data indicated that sh-GRK2 inhibited the expression of TNFα, IL 6 and IL 1β (Figure 2E). Proliferating cell nuclear antigen (PCNA) is a key marker for proliferation, and we evaluated cell proliferation in flap by detecting PCNA expression. IHC staining showed that sh-GRK2 promoted PCNA level (Figure 2F). And deletion of GRK2 accelerated Ki67 expression comparing with NC group upon IR injury (Figure 2G). These data indicated that loss of GRK2 improved skin flap function and cell proliferation after IR injury.
Figure 2.
Knockdown of GRK2 promoted skin function and cell proliferation after IR injury. AAV9-sh-GRK2 or sh-NC was injected into mice through tail vein, then mice were suffered from IR operation after 4 weeks. A. qRT-PCR analysis for GRK2 expression. B. Representative photographs of skin flaps and the survival rate of the total flap area. C. TUNEL for skin flaps to detect apoptotic level. Bar = 100 μm. D. H&E staining was used to determine inflammation level. Bar = 500 μm. E. qRT-PCR analysis for inflammation cytokines TNFα, IL 6 and IL 1β. F. IHC staining for PCNA to determine cell proliferation. Bar = 500 μm. G. The mRNA level of Ki67 was detected using qRT-PCR. n = 10, *P<0.05, **P<0.01 vs sh-GRK2+IR. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Loss of GRK2 increased the number of hair follicles after IR injury
Hair follicles are one of the most important skin appendages and play an important role in wound healing, and we evaluated the effect of sh-GRK2 on hair follicles after IR operation. The H&E images showed that the number of hair follicles was increased in sh-GRK2 group than that in sh-NC group after IR injury (Figure 3A-C).
Figure 3.
Knockdown of GRK2 increased the number of hair follicles after I/R operation. (A) Sections were stained with H&E staining. Bar = 1 mm. (B) Higher magnification of the area inside the rectangle in (A) is shown. Bar = 500 μm. (C) Statistical analysis of hair follicles under HPF. n = 6 for each group. n = 10, *P<0.05 vs sh-GRK2+IR. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Deficiency of GRK2 increased mitochondrial function
Considering the role of GRK2 in modulating mitochondrial function, we measured mitochondrial function in skin after IR injury. Comparing with sh-NC group, mitochondrial ATP production and ATP content were both increased in sh-GRK2 group upon IR operation (Figure 4A and 4B). Knock-down of GRK2 promoted the activities of mitochondrial complexes I, II+III, and IV in comparison with sham (Figure 4C-E). Furthermore, the basal oxygen consumption rate (OCR) and the maximum OCR were increased in sh-GRK2 group upon IR operation than that in sham (Figure 4F and 4G). As well, loss of GRK2 promoted the level of proton leak (Figure 4H). Taken together, these data revealed that deficiency of GRK2 promoted mitochondrial function in skin after IR injury.
Figure 4.
Mitochondrial function in skin flaps after I/R operation. A. Relative mitochondrial ATP production in skin flaps. B. Relative mitochondrial ATP content in skin flaps. C. Relative respiratory chain complex I activity in mitochondria. D. Relative respiratory chain complex II+III activity in mitochondria. E. Relative respiratory chain complex IV activity in mitochondria. The mitochondrial fraction prepared was subjected to Seahorse analyses. F. Basal respiration. G, H. Maximal respiration. OCR indicates oxygen consumption rate. n = 10, *P<0.05 vs sh-GRK2+IR. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Silencing of GRK2 suppressed Drp1 expression in skin after IR injury
Drp1 is a GTPase that regulates mitochondrial fission, and plays key role in mitochondrial function. And we found that the mRNA and protein level of Drp1 were induced in in skin upon IR stress, which was reversed after silencing of GRK2 (Figure 5A and 5B). In addition, IHC staining showed IR injury up-regulated Drp1 expression in flap, while sh-GRK2 down-regulated Drp1 expression (Figure 5C).
Figure 5.
Knockdown of GRK2 inhibited Drp1 expression in skin flap after I/R operation. A. qRT-PCR assay analyzed the expression of Drp1 in skin flaps. B. Western blot detected the protein expression of Drp1 in skin flaps. C. IHC staining for Drp1 in skin flaps. Bar = 500 μm. n = 10, *P<0.05 vs sham, #P<0.05 vs sh-GRK2+IR. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Inhibition of GRK2 improved skin function and mitochondrial function by inhibiting Drp1
To investigate whether Drp1 was involved in GRK2 regulating skin function and mitochondrial function, we constructed AAV9-Drp1 and injected into mice. As shown in Figure 6A, injection of AAV9-Drp1 significantly promoted Drp1 expression in flap. Followed functional experiments showed that overexpression of Drp1 removed the protective effect of sh-GRK2. Comparing with sh-GRK2 group, Drp1 inhibited flap survival rate, promoted apoptosis level, suppressed cell proliferation and induced inflammation in flap upon IR stress (Figure 6B-E). As well, forced expression of Drp1 inhibited the number of hair follicles (Figure 6F). Moreover, sh-GRK2 improved mitochondrial function, which was reversed by Drp1, showing lower level of ATP production and basal OCR (Figure 6G and 6H). Moreover, overexpressing Drp1 alone also aggravated IR-induced flap injury (Figure S1). These results showed that sh-GRK2 improved skin function and mitochondrial function by inhibiting Drp1 expression.
Figure 6.
Overexpression of Drp1 reversed the protective effect of sh-GRK2 in skin flaps after I/R operation. AAV9-sh-GRK2 and AAV9-Drp1or AAV-NC was injected into mice through tail vein, then mice were suffered from IR operation after 4 weeks. A. qRT-PCR for Drp1 expression. B. Representative photographs of skin flaps and the survival rate of the total flap area. C. TUNEL for skin flaps. Bar = 100 μm. D. H&E staining for inflammatory cells. Bar = 500 μm. E. IHC staining for PCNA. Bar = 500 μm. F. H&E staining for hair follicles under HPF. Bar = 500 μm. G. Relative mitochondrial ATP production in skin flaps. H. Basal OCR of skin flaps. n = 10, *P<0.05 vs sh-GRK2+IR+NC. Data among multiple groups were analyzed by one-way ANOVA, followed by a Tukey post hoc test. The experiment was repeated in triplicate.
Discussion
Ischemia-reperfusion is one of the main factors leading to the necrosis of skin flap after transplantation [15]. The ischemia time of the flap is closely related to the reperfusion injury [16]. Therefore, surgeons are required to improve their own operation techniques and shorten the operation time. In addition, it is more important to actively seek drugs and other treatment methods to maintain the activity and function of skin cells and reduce the occurrence of ischemia-reperfusion injury of skin flap.
GRK2 is widely distributed in various tissues and organs, and it regulates the body’s pathophysiological processes by phosphorylating related signaling pathways involved in GPCRs and non-GPCRs pathways [17]. GRK2 is closely related to the functions of multiple organs such as heart, liver, kidney and lung [18]. It has been reported that GRK2 plays a key role in IR injury of heart, and deletion of GRK2 enhanced cardiac function [19]. And in present study, we constructed an IR model of mice abdominal flap. In IR group, the skin flaps were brown and inelastic, and had a high apoptotic level. And the Comparing with sham group, GRK2 was increased in flap after IR injury. To explore the role of GRK2 in skin flap after IR injury, we injected AAV9-sh-GRK2 to inhibit GRK2 expression in mice. A normal proliferative ability of skin cell is necessary for repair after IR injury [20]. Further, hair follicles are appendages of skin, which reflect the secretory function of the skin [21]. And present study found that loss of GRK2 inhibited cell apoptosis, promoted cell proliferation of flap and increased the number of hair follicles after IR injury.
At present, it is believed that the resumption of blood supply of tissues and organs after a long period of ischemia can lead to the outbreak of ROS, the change of mitochondrial function, and eventually leads to tissue necrosis [22]. Excessive ROS in tissues will enhance lipid peroxidation and change the lipid microenvironment of ion channels, cell membrane receptors and proteases, thus damaging cell functions [23]. Oxygen free radicals can also cause vasoconstriction, increased capillary permeability, swelling in the endothelial cell, and damaged skin flap microcirculation process, causing necrosis of the organization [24]. As the main place of energy metabolism in eukaryotes, mitochondria play an important role in ROS production, further mitochondria are also key target organelles for oxidative stress [25]. Interestingly, our study showed that deletion of GRK2 improved mitochondria function in skin flaps after IR injury.
Drp1 is the main protein that mediates mitochondrial division [26]. The fusion and fission of mitochondria are the basic events in the cell, and maintaining the dynamic balance between them is very important for the normal morphology [27]. In recent years, it has been found that IR leads to the change of mitochondrial dynamics from fusion to division in heart, and the inhibition of excessive mitochondrial division is beneficial to the resistance to IR injury in heart [28]. Numerous studies indicate the mitochondrial division mediated by Drp1 is involved in IR injury [29]. IR disordered muscle fiber structure and increased mitochondrial fragmentation, while silencing of Drp1 significantly reduced the myocardial infarction area and improved cardiac function [30]. And our study showed that IR injury up-regulated Drp1 expression in skin flaps, while sh-GRK2 down-regulated Drp1 expression. Furthermore, overexpression of Drp1 removed the protective effect of sh-GRK2, which indicated that deletion of GRK2 improved skin flap function through inhibiting Drp1 in ischemia-reperfusion injury.
Conclusion
In conclusion, our study revealed that GRK2 deletion improved flap function and mitochondrial function by inhibiting Drp1 expression, which may provide a new insight for the clinical treatment of flap ischemia-reperfusion injury.
Acknowledgements
This work were supported by the Wuxi Health Management Committee, Grant No. Q201827 and LCZXJS001.
Disclosure of conflict of interest
None.
Supporting Information
References
- 1.Inagaki T, Morino T, Takagi R, Yamato M, Koizuka I, Yaguchi Y. Transplantation of autologous oral mucosal epithelial cell sheets inhibits the development of acquired external auditory canal atresia in a rabbit model. Acta Biomater. 2020;110:141–152. doi: 10.1016/j.actbio.2020.04.031. [DOI] [PubMed] [Google Scholar]
- 2.Pu CM, Liu CW, Liang CJ, Yen YH, Chen SH, Jiang-Shieh YF, Chien CL, Chen YC, Chen YL. Adipose-derived stem cells protect skin flaps against ischemia/reperfusion injury via IL-6 expression. J Invest Dermatol. 2017;137:1353–1362. doi: 10.1016/j.jid.2016.12.030. [DOI] [PubMed] [Google Scholar]
- 3.Lee JH, Suh JH, Kang HJ, Choi SY, Jung SW, Lee-Kwon W, Park SA, Kim H, Ye BJ, Yoo EJ, Jeong GW, Park NH, Kwon HM. Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance. EBioMedicine. 2020;58:102926. doi: 10.1016/j.ebiom.2020.102926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Edmunds MC, Czopek A, Wigmore SJ, Kluth DC. Paradoxical effects of heme arginate on survival of myocutaneous flaps. Am J Physiol Regul Integr Comp Physiol. 2014;306:R10–22. doi: 10.1152/ajpregu.00240.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.San Juan-Reyes S, Gomez-Olivan LM, Islas-Flores H, Dublan-Garcia O. Oxidative stress in pregnancy complicated by preeclampsia. Arch Biochem Biophys. 2020;681:108255. doi: 10.1016/j.abb.2020.108255. [DOI] [PubMed] [Google Scholar]
- 6.Angel MF, Im MJ, Chung HK, Vander Kolk CA, Manson PN. Effects of combined cold and hyperbaric oxygen storage on free flap survival. Microsurgery. 1994;15:648–651. doi: 10.1002/micr.1920150909. [DOI] [PubMed] [Google Scholar]
- 7.Nakai K, Kadiiska MB, Jiang JJ, Stadler K, Mason RP. Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin. Proc Natl Acad Sci U S A. 2006;103:4616–4621. doi: 10.1073/pnas.0510352103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Arumugam S, Girish Subbiah K, Kemparaju K, Thirunavukkarasu C. Neutrophil extracellular traps in acrolein promoted hepatic ischemia reperfusion injury: therapeutic potential of NOX2 and p38MAPK inhibitors. J Cell Physiol. 2018;233:3244–3261. doi: 10.1002/jcp.26167. [DOI] [PubMed] [Google Scholar]
- 9.Chen G, Shen H, Zang L, Su Z, Huang J, Sun Y, Wang H. Protective effect of luteolin on skin ischemia-reperfusion injury through an AKT-dependent mechanism. Int J Mol Med. 2018;42:3073–3082. doi: 10.3892/ijmm.2018.3915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gurevich VV, Gurevich EV. Biased GPCR signaling: possible mechanisms and inherent limitations. Pharmacol Ther. 2020;211:107540. doi: 10.1016/j.pharmthera.2020.107540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ciccarelli M, Sorriento D, Fiordelisi A, Gambardella J, Franco A, Del Giudice C, Sala M, Monti MG, Bertamino A, Campiglia P, Oliveti M, Poggio P, Trinchese G, Cavaliere G, Cipolletta E, Mollica MP, Bonaduce D, Trimarco B, Iaccarino G. Pharmacological inhibition of GRK2 improves cardiac metabolism and function in experimental heart failure. ESC Heart Fail. 2020;7:1571–1584. doi: 10.1002/ehf2.12706. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Liu R, Li X, Zhu W, Wang Y, Zhao D, Wang X, Gurley EC, Liang G, Chen W, Lai G, Pandak WM, Robert Lippman H, Bajaj JS, Hylemon PB, Zhou H. Cholangiocyte-derived exosomal long noncoding RNA H19 promotes hepatic stellate cell activation and cholestatic liver fibrosis. Hepatology. 2019;70:1317–1335. doi: 10.1002/hep.30662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Sato PY, Chuprun JK, Ibetti J, Cannavo A, Drosatos K, Elrod JW, Koch WJ. GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels. J Mol Cell Cardiol. 2015;89:360–364. doi: 10.1016/j.yjmcc.2015.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14. doi: 10.1038/nrm3028. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Schuster R, Bar-Nathan O, Tiosano A, Lewis EC, Silberstein E. Enhanced survival and accelerated perfusion of skin flap to recipient site following administration of human alpha1-antitrypsin in murine models. Adv Wound Care (New Rochelle) 2019;8:281–290. doi: 10.1089/wound.2018.0889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Perrault DP, Lee GK, Bouz A, Sung C, Yu R, Pourmoussa AJ, Park SY, Kim GH, Jiao W, Patel KM, Hong YK, Wong AK. Ischemia and reperfusion injury in superficial inferior epigastric artery-based vascularized lymph node flaps. PLoS One. 2020;15:e0227599. doi: 10.1371/journal.pone.0227599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Penela P, Ribas C, Sanchez-Madrid F, Mayor F Jr. G protein-coupled receptor kinase 2 (GRK2) as a multifunctional signaling hub. Cell Mol Life Sci. 2019;76:4423–4446. doi: 10.1007/s00018-019-03274-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Eibel B, Kristochek M, Peres TR, Dias LD, Dartora DR, Casali KR, Kalil RAK, Lehnen AM, Irigoyen MC, Markoski MM. beta-blockers interfere with cell homing receptors and regulatory proteins in a model of spontaneously hypertensive rats. Cardiovasc Ther. 2018;36:e12434. doi: 10.1111/1755-5922.12434. [DOI] [PubMed] [Google Scholar]
- 19.Woodall MC, Woodall BP, Gao E, Yuan A, Koch WJ. Cardiac fibroblast GRK2 deletion enhances contractility and remodeling following ischemia/reperfusion injury. Circ Res. 2016;119:1116–1127. doi: 10.1161/CIRCRESAHA.116.309538. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Pu CM, Chen YC, Chen YC, Lee TL, Peng YS, Chen SH, Yen YH, Chien CL, Hsieh JH, Chen YL. Interleukin-6 from adipose-derived stem cells promotes tissue repair by the increase of cell proliferation and hair follicles in ischemia/reperfusion-treated skin flaps. Mediators Inflamm. 2019;2019:2343867. doi: 10.1155/2019/2343867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Migda MS, Migda M, Slapa R, Mlosek RK, Migda B. The use of high-frequency ultrasonography in the assessment of selected female reproductive structures: the vulva, vagina and cervix. J Ultrason. 2019;19:261–268. doi: 10.15557/JoU.2019.0039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Zheng K, Zhang Q, Sheng Z, Li Y, Lu HH. Ciliary neurotrophic factor (CNTF) protects myocardial cells from oxygen glucose deprivation (ogd)/re-oxygenation via activation of Akt-Nrf2 signaling. Cell Physiol Biochem. 2018;51:1852–1862. doi: 10.1159/000495711. [DOI] [PubMed] [Google Scholar]
- 23.Zamarron A, Morel E, Lucena SR, Mataix M, Perez-Davo A, Parrado C, Gonzalez S. Extract of deschampsia antarctica (EDA) prevents dermal cell damage induced by uv radiation and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int J Mol Sci. 2019;20:1356. doi: 10.3390/ijms20061356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Guo-Qian Y, Gang W, Zhi-Yong S. Investigation on the microcirculation effect of local application of natural hirudin on porcine random skin flap venous congestion. Cell Biochem Biophys. 2012;62:141–146. doi: 10.1007/s12013-011-9274-5. [DOI] [PubMed] [Google Scholar]
- 25.Heimbucher T, Qi W, Baumeister R. TORC2-SGK-1 signaling integrates external signals to regulate autophagic turnover of mitochondria via mtROS. Autophagy. 2020;16:1154–1156. doi: 10.1080/15548627.2020.1749368. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Yu B, Ma J, Li J, Wang D, Wang Z, Wang S. Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics. Nat Commun. 2020;11:2549. doi: 10.1038/s41467-020-16312-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ali L, Haynes CM. Mitochondrial translation, dynamics, and lysosomes combine to extend lifespan. J Cell Biol. 2020;219:e202005084. doi: 10.1083/jcb.202005084. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Cheng QQ, Wan YW, Yang WM, Tian MH, Wang YC, He HY, Zhang WD, Liu X. Gastrodin protects H9c2 cardiomyocytes against oxidative injury by ameliorating imbalanced mitochondrial dynamics and mitochondrial dysfunction. Acta Pharmacol Sin. 2020;41:1314–1327. doi: 10.1038/s41401-020-0382-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Han F, Huang D, Huang X, Wang W, Yang S, Chen S. Exosomal microRNA-26b-5p down-regulates ATF2 to enhance radiosensitivity of lung adenocarcinoma cells. J Cell Mol Med. 2020;24:7730–7742. doi: 10.1111/jcmm.15402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Zepeda R, Kuzmicic J, Parra V, Troncoso R, Pennanen C, Riquelme JA, Pedrozo Z, Chiong M, Sanchez G, Lavandero S. Drp1 loss-of-function reduces cardiomyocyte oxygen dependence protecting the heart from ischemia-reperfusion injury. J Cardiovasc Pharmacol. 2014;63:477–487. doi: 10.1097/FJC.0000000000000071. [DOI] [PubMed] [Google Scholar]
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