Secretion of growth factors

Secretion of implanted cells plays an important role in repairing heart tissue damage. Adult stem cells, particularly MSCs, after transplantation can release a variety of cytokines, chemokines and growth factors involved in heart repair process Deb et al., 2008Li et al., 2012Loffredo et al., 2011. These factors, in turn, induce neighboring stem cells to secrete cytokines and induce changes in the microenvironment which promote proliferation and differentiation of stem cells Behfar et al., 2002Doyle et al., 2016Gude et al., 2015Kinnaird et al., 2004. In particular, properties such as myocardial protection and neovascularization of paracrine factors currently have been most widely studied. Moreover, the secreted factors also impact positively on the inflammation process, fibrogenic process, heart metabolism, heart contractions and/or endogenous cardiac regeneration. These effects may occur in different ways and are dependent on the microenvironment after infarction. These factors may also act in an autocrine fashion, impacting the cells which secrete them Deb et al., 2008.

Protection of heart tissue

The immediate impact of stem cells after grafting into the heart muscle is to release cytoprotective molecules to increase myocardial viability. These molecules inhibit apoptosis by activating AKT/PKB signaling pathway (Rosenberg et al., 2012; Yang et al., 2012). Some studies have shown that Aktoverexpressing MSCs and exosomes secreted from CXCR4-overexpressing or GATA4-overexpressing MSCs are able to significantly prevent apoptosis, thereby reducing infarction size Kang et al., 2015Noiseux et al., 2006Yu et al., 2015. Other studies have shown that elements secreted from grafted bone marrow stem cells induce cardiomyocyte protection in the ischemic region Broughton and Sussman, 2016Dai et al., 2008Xu et al., 2007. Cardiomyocytes differentiated from bone marrow monocytes (BM-MNCs) cultured under hypoxic conditions show they have inhibitory effects on apoptosis and can reduce infarction size when transplanted into the body Kubal et al., 2006. When heart muscle cells and BM-MNCs obtained from the same patients were co-cultured, cell necrosis and apoptosis were significantly reduced; cell protection, however, did not occur when heart muscle cells with co-cultured with ECs or keratinocytes Yoon et al., 2005.

Moreover, transplanted Akt-expressing MSCs also expressed secreted frizzled related protein 2 (Sfrp2), which increases cellular total -catenin of cardiomyocytes. The -catenin protected cardiomyocytes of newborn rats were stable against hypoxia and reoxygenation-induced apoptosis by blocking the pre-apoptotic effects of Wnt3a Mirotsou et al., 2007Zhang et al., 2009. AktMSCs regulated the increase of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and thymosin 4 (TB4). In turn, these factors promoted neovascularization, protection of myocardium and cardiac contractility Gnecchi et al., 2006 ( Figure 2 ).

The overexpression of bFGF can enhance the recovery of contractile function and reduce infarction area after reperfusion. In fact, bFGF reduces the protein kinase C delta (PKC-d) displacements but does not affect PKC-alpha (PKC-a), PKC-epsilon (PKC-e), or PKC-zeta (PKC-z). PKC-d reduction protects heart cells and decreases the number of dead cells. In addition, bFGF has been shown to be related to the MAPK/ERK signaling pathway in heart cell protection, although the mechanism remains unclear Baines et al., 2002House et al., 2007Padua et al., 1998Rose et al., 2010Srisakuldee et al., 2014. Akt activates a number of substrates, including members of the B-cell lymphoma 2 (Bcl-2) protein family, glycogen synthase kinase 3 beta (GSK-3) and endothelial nitric oxide synthase (eNOS). Nitric oxide (NO) synthesized from eNOS activates PKG through intracellular cGMP increasing. The substrates for protein kinase G (PKG) are thought to include SR regulation proteins and phospholamban, which promotes SR Ca absorption thereby reducing the overload of cytosolic Ca. PKG is the final component of the signal transduction leading to activation of PKC-epsilon (PKC-e) mitochondrial pool. Activated PKC-e in turn activates mitochondrial

ATP-dependent potassium channels (mK), promoting the reactive oxygen species (ROS) formation ( Figure 2 ).

The inhibition of mitochondrial permeability transition pore (MPTP) may occur as a result of PKC-e activation. Sarcolemmal K (sK) and mitochondrial connexin-43 (Cx43) are also considered components of the pre-regulation mechanism. The formation of ROS and reactive nitrogen species (RNS) are the results of mK opening and are mandatory components of the signaling cascade. It seems that ROS /RNS signaling is related to activation of kinases, such as p38, MAPK, PKC and JAK/STAT Ferdinandy et al., 2007.

The heart protects extracellular ligands, including adenosine, bradykinin and opioids, while activating multiple kinases, including p42/p44, MAPK/ERK, PI3K/Akt and PKC. The substrates of PKC related to the regulating protective mechanisms are still unknown. There is also evidence that the activation of PKG might occur as part of the post-regulating mechanism downstream of Akt/NO/cGMP, and may play a role in promoting the absorption of sarcoplasmic reticulum Ca. It is known that activation of Akt inhibits GSK-3 and members of the chemokine (C-C motif) ligand 2 (Ccl-2) protein family, leading to inhibition of the formation of mKmediated MPTP Ferdinandy et al., 2007.

Normally, PKC exists in a non-activated state until it is stimulated by phospholipid diacyl glycerol (DAG)-derived second messengers. Intracellular ROS translocates to the subcellular target locations, including mitochondria, sarcolemmal membrane and gap junctions. Indeed, sKmediated phosphorylation leads to shortening of the action potential, reducing the Ca overload during ischemia. Moreover, PKC-mediated phosphorylation of Cx43 reduces connexon permeability and prevents the expansion of damages between co-joined cells. PKC-mediated phosphorylation opens the mK, conserves mitochondria function and forms local ROS. In turn, ROS may initiate PKC signaling via positive feedback. MPTP inhibition can occur by both direct mechanisms (e.g. via PKC phosphorylation) and indirect mechanisms (e.g. via mK opening which reduces cell death after infarction). Receptor for activated c-kinase (RACK) treatment and erythropoietin also promote PKC activation, resulting in heart cell protection Bearzi et al., 2007Budas et al., 2007.

In summary, during ischemia reperfusion activated PKC-e in turn activates sK and mK, phosphorylates Cx43 and inhibits MPTP, leading to cell protection. However, activated PKC-d inhibits pyruvate dehydrogenase (PDH), decreases Bcl-2, increases Bcl-2-associated death promoter (Bad) protein, causing apoptosis and necrosis. Thus, transplanted cells protect myocardium by secreting various factors that activate PKC-e and inhibit PKC-d.

Mobilization of resident stem cells

The discovery and recognition of the existence of cardiac stem cells led to a shift by researchers and physicians to explore the use of stem cells for new cardiovascular disease therapies Bearzi et al., 2009Hosoda et al., 2009Urbanek et al., 2006. In the heart, cardiac stem cells (CSCs) have existed in their niches. Normally, these cells have kept "silent" in their niches, and have been nourished and controlled by feeding cells within the niches. Upon receiving trigger signals, CSCs undergo a symmetrical or asymmetrical proliferation; they separate from their niches and migrate to areas where they were needed to replace damaged or dying heart cells. However, the number of CSCs are very rare, at approximately 1 stem cell per 30,000 heart cells Beltrami et al., 2003Urbanek et al., 2006. Therefore, based on the normal growth rate of resident CSCs, when infarction occurs the number of cells needed to be replaced is much greater than the number of available cells, leading to a lack of intrinsic cardiac stem cells for replacement. This means that while the injured heart may be delayed in damages in the short term, heart failure cannot be reversed in the long term Urbanek et al., 2003.

Studies have shown that when MSCs are grafted into the body in the absence of oxygen, they release HGF and IGF-1 to mobilize and activate resident CSCs Gómez-Mauricio et al., 2016Linke et al., 2005. Besides MSCs, endothelial progenitor cells (EPCs) play an important role. EPCs activate cardiac regenerative pools and promote the migration, proliferation and differentiation of CSCs (via secretion of cytokines such as VEGF, IGF-1 and SDF-1) Urbich et al., 2005. These factors induce interstitial CSCs to move through the myocardium to necrotic myocardium and scar areas. There, they divide and differentiate into heart cells and become involved in the process of new blood vessel formation Bian et al., 2014Hosoda et al., 2009Tillmanns et al., 2008. CSC invasion to scar tissue is believed to be related to matrix metalloproteinase (MMP)-9 and -14 mediated regulation Bax et al., 2012Huang et al., 2011Rota et al., 2008.

Neovascularization

MSC transplantation improves reperfusion efficiency by increasing the formation of new blood vessels. However, MSCs are rarely present in the new vessels; their main activity is to secrete angiogenic factors, such as VEGF, bFGF, angiopoietin-1, NO and HGF Kinnaird et al., 2004Zhao et al., 2010. These factors increase the permeability of the capillary wall, activate MMP, promote the proliferation and migration of ECs and vascular smooth muscle cells (VSMCs), and form new vessels in the lesions Jiang et al., 2006Louis and Zahradka, 2010Nagaya et al., 2004. Besides MSCs, transplanted EPCs also enhance new vessel formation by releasing VEGF and stromal cell-derived factor 1 (SDF-1) into

the cellular matrix, thereby promoting the migration and maturation of EPCs into ECs Cochain et al., 2013Urbich et al., 2005Wu et al., 2006. Angiogenic cells have been implanted as hydrogel supplying scaffolds to increase microvasculature along infarction areas, thereby significantly improving coronary blood flow and ejection fraction after MI Kim et al., 2012Leblanc et al., 2013Levit et al., 2013. In addition to angiogenesis factors, adult stem cells secrete TB4 and erythropoietin (EPO) Lv et al., 2015Smart et al., 2007. TB4 induces

the proliferation and circuit network formation of epicardium-derived cells (EPDCs) and is involved in the intermediate PKC signaling pathway Smart et al., 2013Smart et al., 2007. Meanwhile, granulocyte colony stimulating factor (GCSF) and EPO mobilize hematopoietic stem cells (HSCs) and EPCs from bone marrow for angiogenesis by activating Janus-activated tyrosine kinase 2 (JAK2) through STAT, PI3K/Akt and MAPK signaling pathways (Nagai and Komuro, 2012).

Impact on extracellular matrix (ECM) and reduction of scar formation

After infarction, scars are formed to replace injury tissues damaged by myocardial ischemia. During ischemia, the ECM secretion process is disordered due to heart cells dying and the body's self-regulation. These impact the thickness of the developing scar, leading to an effect on the contraction of the surrounding heart tissue. A decrease in the ECM makes the ventricular wall thinner, causing left ventricular (LV) rupture while an increase in the ECM enhances fibrosis, leading to heart failure over time Zamilpa and Lindsey, 2010.

From studies, it has been observed that grafted cells are capable of regulating scar formation through inhibition of fibroblast proliferation; furthermore, it has been shown that paracrine factor secretion alters ECM to improve cardiac functions Berry et al., 2006. Transplanted MSCs reduce myofibroblasts through releasing MMPs Almalki and Agrawal, 2016Mias et al., 2009. In MI rat models, implanted MSCs reduce the expression of collagen types I and III, tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP2, MMP9, and transforming growth factor-beta (TGF-beta) Nagaya et al., 2005Xu et al., 2005. It is interesting that transplanted MSC cardiomyocyte (MSC-CM) also express the ability to reduce the scarring process by downregulating fibroblast proliferation and inhibiting the expression of collagen type I and type III in myofibroblasts Ohnishi et al., 2007aOhnishi et al., 2007b. In addition, embryonic stem cell cardiomyocyte (ESC-CM) also show the ability to reduce scarring after MI Leor et al., 2007. In sheep, MSCs injected one hour after MI also show changes in MMP-1, -2, -3, -7, -9, -13, membrane type 1-MMP (MT1-MMP), and TIMPs-1, -2, -4, at the border zone and infarct zone Dixon et al., 2009.

Limiting inflammation

After MI, the inflammatory process is needed to mobilize immune cells to clear out dead heart cells and debris, and to stimulate ventricular remodeling Frangogiannis, 2012Frangogiannis et al., 2002. However, prolonged inflammatory responses would be detrimental to the remodeling process and ventricular function due to heart cell loss leading to negative impacts on ECM as well as formation of new vessels Frangogiannis et al., 2002. Transplanted cells, such as MSCs or MSC-CMs, are able to limit the inflammatory process in the injured tissue. They weaken the proliferation of inflammatory CD68+ cells, decrease the expression of monocyte chemoattractant protein (MCP-1), increase the expression of genes involved in DNA repair, increase antioxidant enzymes and stimulate detoxifier systems, thereby improving cardiac function Fuse et al., 2001Ohnishi et al., 2007bRamalho-Santos et al., 2002.

Transplanted MSCs increase the number of M2 macrophages which were at the anti-inflammatory stage. The mechanism of this process is related to a variety of paracrine factors derived from transplanted MSCs, such as CCL2, galectin-1, interferon-γ, interleukin (IL)-1β, indoleamine-2,3-dioxygenase, IL-4, IL-6, IL-10, IL-13, prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), NO, heme oxygenase-1, HGF, and TGF-beta Ben-Mordechai et al., 2013Bernardo and Fibbe, 2013Du et al., 2008. Normally, the pro-inflammatory cytokines (e.g. NF-κB, IL-6 and TNF-) poison the heart muscle cells, causing detrimental effects on cardiac contractile function. Transplanted MSCs have been shown to inhibit activation of NF-κB, reduce production of TNF- α and IL-6, and increase the expression of anti-inflammatory cytokine IL-10, thereby limiting inflammation after infarction Du et al., 2008Onai et al., 2007. Moreover, the increase of indoleaminepyrrole 2,3-dioxygenase (IDO) and PGE2 secretion also reduces T-cell activity and NK cell proliferation Glennie et al., 2005Nauta et al., 2006Pradier et al., 2011van den Akker et al., 2013.