C1q/TNF-Related Protein-3, A Newly Identified Adipokine, Is A Novel Pro-angiogenic Molecule And Mechanisms Involved

Backgrounds:Due to increased incidence of diabetes and its associationwith heart failure, chronic wound healing and many other diseases, there is nowgreat interest in elucidating the underlying molecular mechanisms linking thesepathologies. Since their original discovery, the important regulatory role ofadipose-derived hormones and cytokines (adipokines) in myocardial functionhas gained recognition. Considerable evidence exists that the majority ofadipokines (including TNF, leptin, plasminogen activator inhibitor type1,transforming growth factor β, and resistin) adversely regulate myocardialmetabolism, cardiomyocyte hypertrophy, extracellular matrix structure andcomposition, and cell death. Despite having been consistently shown to increaseglucose uptake, stimulate fatty acid oxidation, and protect against ischemia/reperfusion injury, adiponectin’s effect upon chronic cardiacremodeling and heart failure development remains controversial. Whether thereexists any adipokine exerting beneficial regulatory effect upon post-ischemiccardiac remodeling, improving cardiac function, remains unknown. Whetherthere is any adipokine enhancing angiogenesis and improving wound healing isstill unclear.Recently, a highly conserved family of adiponectin paralogs, designated C1qtumor necrosis factor (TNF) related proteins (CTRPs), has been discovered.Each of the known fifteen members (CTRP1through15,8and10being humanspecific) consist of four distinct domains, including an N-terminal signal peptide,a short variable domain, a collagen-like domain, and a C-terminal C1q-likeglobular domain. Both CTRPs and adiponectin belong to the C1q/TNF proteinsuperfamily, which continues to grow as more C1Q domain proteins arediscovered. Investigated for its structural similarity to adiponectin, the CTRPfamily members exhibit broadly diverse functions. CTRP3is the first and onlyCTRP whose in vivo biological function (as a metabolic regulator of glucosehomeostasis) has been recently established.Interestingly, CTRP3has been reported to stimulate in vitro endothelial cellproliferation and migration. However, whether CTRP3might promote in vivorevascularization is unknown. Moreover, whether CTRP3, a key member of thenewest adipokine family, may function as a mediator or inhibitor ofpost-ischemic remodeling and wound healing has never been previouslyinvestigated.Objective:1) To determine the effect of CTRP3upon angiogenesis in vitro;2)To determine whether CTRP3administration might promote revascularizationafter myocardial infarction in vivo and the mechanisms responsible for CTRP3’s cardiac biological actions; and3) To determine whether CTRP3administrationmight enhance revascularization in the skin wound healing process.Methods: Construct and expression of globular CTRP3: Globulardomain of mouse CTRP3gene was generated by PCR, and cloned into theprokaryotic protein expression vector pET45b. The construct was verified byDNA sequencing. In vitro experiment:①Adult mouse cardiomyocyteculture: Adult mouse cardiomyocytes were isolated from WT and were serumstarved. Akt inhibitor LY294002was administered15minutes prior to CTRP3treatment in an effort to elucidate the mechanism of CTRP3inducedangiogenesis.②HUVECs were purchased from ATCC.③Tube formation assay:HUVECs were seeded onto Matrigel, in endothelial cell basal medium-2withEGM-2Bullet Kit, and switched to cardiomyocyte-conditioned medium. After6hours, tube length was quantified via Image-Pro Plus5.0. VEGF receptorantagonist CBO-P11was utilized for inhibition studies.④Determination of NOproduction: CTRP3-stimulated nitric oxide production in HUVECs wasdetermined and the amount of NO released was expressed in nmol/mg protein.⑤Western blot analysis: Proteins of interest were separated on SDS-PAGE gels,transferred to PVDF membranes, and incubated with appropriate antibodies.Myocardial infarction (MI) model:①MI was induced via LAD ligation.After full recovery from cardiac surgery, animals were randomized to one of thefollowing groups: Sham+Vehicle (Veh), Sham+CTRP3, MI+Veh, andMI+CTRP3. After2weeks, mice were sacrificed. Immunohistological,biochemical, and Western blot assays were performed upon AAR (area at risk)tissue.②Determination of cardiac function: Cardiac function was determinedby two separate methods:1) echocardiography2) left ventricular catheterization.Determination of cardiac function occurred2weeks after mini-osmotic pump implantation, prior to thoracotomy.③Histological analysis：Cardiac collagencontent and infarct size were assessed after Masson trichrome staining. Cardiacsections were immunohistochemically stained utilizing primary antibodies toCD31, actinin, and α-SMA.④Western blot analysis：Proteins of interest wereseparated on SDS-PAGE gels, transferred to PVDF membranes, and incubatedwith appropriate antibodies. Wound healing model:①The excisionalwounds were made into the dorsal skin of the mice with a6-mm biopsy punch.A total2μg/kg/d CTRP3was injected in the area surrounding a wound. After7days, the mice were sacrificed.②Wound closure and angiogenesis in mouseskin after injection of CTRP3were calculated.③HE staining was performed tomeasure the length and area of new blood vessels.④Staining was performed in4μm paraffin tissue sections to assess the expression of CD31andαSMA. Statistical analysis: All values in the text and figures are presented asthe mean±SEM of n independent experiments. ANOVA was conducted acrossall investigated groups first. Post hoc pairwise tests for certain group pairs, withassessment of statistical significance, were then performed after Bonferronicorrection of the overall significance level.Results: CTRP3does not significantly promote angiogenesis incultured HUVECs. Direct HUVECs treatment with full length or globulardomain of CTRP3failed to significantly promote tube formation or stimulateNO production. Moreover, although in vivo CTRP3treatment significantlyincreased Akt phosphorylation and increased HIF1-α and VEGF expression inthe ischemic heart, in vitro treatment of HUVECs with CTRP3neither increasedAkt phosphorylation, nor HIF1-and VEGF expression. CTRP3promotesangiogenesis via a cardiomyocyte-mediated, Akt-dependent pathway.HUVECs treatment with conditioned medium from cardiomyocyte significantly enhanced HUVECs tube formation, suggesting that CTRP3invokes anangiogenic response by inducing cardiomyocyte-secreted paracrine factors.Furthermore, CTRP3up-regulated levels of pAkt, HIF-1, and VEGF incultured cardiomyocytes in both time-and dose-dependent manner. Additionally,blocking cardiomyocyte Akt activation abolished CTRP3-induced HIF-1ɑ andVEGF up-regulation, and eliminated HUVECs tube formation induced by theconditioned medium. Moreover, HUVECs tube formation induced by theconditioned medium from CTRP3-treated cardiomyocytes was significantlysuppressed by a VEGF-specific inhibitor administered to HUVECs. Exogenous CTRP3improves survival and restores left ventricular cardiacfunction after MI. CTRP3administration significantly improved post-MIsurvival rate (P<0.05), augmented left ventricular ejection fraction (LVEF) by57.0%, increased dP/dtmax by30.4%, and decreased LV end diastolic pressure(LVEDP) by17.6%. CTRP3prevents left ventricular cardiac remodelingafter MI. We determined indices of remodeling by gross anatomy,echocardiography, and Masson’s trichrome staining14days after MI. CTRP3administration reduced cardiac size and mass and preserved LV end-diastolicdimension (LVEDD) and LV end-systolic dimension (LVESD). Most noticeably,CTRP3treatment significantly attenuated interstitial fibrosis in the infarctborder zone (41%reduction vs. MI+Veh), and dramatically increasing themuscular cell/fibrotic cell ratio in the ischemic region (P<0.01). CTRP3promotes angiogenesis and activates the Akt-HIF1α-VEGF axis in infarctborder zone. We assessed the potential angiogenic effects of CTRP3via CD31immunohistochemical staining. CTRP3treatment markedly increased thenumber of CD31-positive capillary vessels and-SMA–positive arterial densityin the border zone14days after MI. CTRP3had no significant effect upon AMPK or eNOS phosphorylation, but significantly enhanced Aktphosphorylation, and increased HIF1-α and VEGF expression, suggesting thepossible involvement of the Akt-HIF1α-VEGF axis in this process. Finally, toconfirm whether CTRP3cardioprotection is through Akt-VEGF axis, continuousinhibition of Akt or VEGF via mini-osmotic pump was performed post MI, andcardiac function and capillary density were determined in vivo. Compared toMI+CTRP3, inhibition of Akt largely blocked CTRP3’s restore of LVEF andalmost eliminated CTRP3’s promotion of angiogenesis. However, althoughinhibition of VEGF prevented CTRP3’s angiogenesis effect at same levelcomparing to inhibition of Akt, it only blunted partial of cardioprotective effectof CTRP3on cardiac function. CTRP3significantly inhibited infarctborder zone cardiomyocyte apoptosis. CTRP3treatment significantly reducedapoptotic cell death, as evidenced by decreased TUNEL-positive staining andreduced caspase-3activation. Finally, although CTRP3-KO mouse isunavailable, we utilized conditioned medium of induced3T3cell line andco-cultured with isolated adult mouse cardiomyocyte to determine whetherendogenous CTRP3may play important role in regulating cardioprotectionagainst ischemic injury. Specific SiRNA of CTRP3or scramble (none relatedSiRNA) were utilized to3T3cells48hours before co-cultured with adultcardiomyocytes. Then adult cardiomyocytes were exposed to simulatedischemia (SI,2%O2for12hours) and LDH release and Caspase3activity weredetermined at the end point. Comparing with SI only, pretreated with3T3-cellssignificantly attenuated cell death and apoptotic events. Moreover, inhibition ofCTRP3in3T3-cells at least partially blunted this beneficial effect against SIinjury in vitro. CTRP3accelerates wound healing by promotingangiogenesis in mice. CTRP3significantly enhanced wound closure as early as 3days after injury and with a30%decrease in wound size7days after injury.The number of blood vessels in the subcutaneoustissue surrounding the woundof the CTRP3groupwas significantly higher than in the control groupasevaluated from photos, by HE staining and αSMAimmunostaining.Conclusions:Our study presents several important observations: To ourbest knowledge, we demonstrate for the first time that CTRP3, a key member ofa newly identified adipokine family, exerts its angiogenic effect by promotingcell-cell communication; CTRP3improves survival and restores cardiacfunction after MI induction in a mouse model through enhancing infarct borderzone revascularization and reducing apoptosis; CTRP3promotesangiogenesis around the border zone after myocardial infarction involvingAkt-HIF1-VEGF signaling activation； and CTRP3accelerates woundhealing via increasing angiogenesis around the wound.