Molecular Mechanisms of Cell Protection Against Injury—The Foundation of Cell Protective Engineering

A cell is a dynamic biological system capable of sensing and adapting environmental changes to suit its ability to survive and function [1]. In the event of exposure to an injury-inducing environmental insult, such as ischemia, mechanical impact, chemical toxicity, or microorganism infection, a cell can activate its protective mechanisms to reduce the possibility of death, a defense function established during evolution through repeated interactions with environmental insults [1,2]. The cell protective function is carried out by various cell-survival supporting systems, consisting of extracellular stimulatory ligands released from regionally injured and activated cells as well as recruited distant cells, cell membrane receptors, intracellular signaling cascades, and genes [1–7]. Although the cell-survival supporting systems can be activated in response to an environmental insult, the protective processes are not always optimized in the timing of activation and the level of effectiveness—the actions of most cell-survival systems often take place after cell death with an insufficient level of impact. These problems can be potentially addressed by controlled modifications of selected cell-survival systems—an approach known as Cell Protective Engineering [1,2]. Established protective engineering strategies include protein-level modifications by controlled protein delivery, gene transfer to promote gene expression, mRNA interference to knock down the levels of mRNAs that impede cell protection, gene editing to modify the level of gene expression, and/or cell transplantation to regionally release protective factors to the injury site. The application of adequately selected engineering strategies to injured cells can lead to better-controlled timing and levels of cell protective actions, thereby maximizing the capacity of cell survival in the event of injury. In this article, experimental myocardial ischemia is used as a model to demonstrate the molecular mechanisms of cell protection against injury as well as the concept of cell protective engineering established based on the naturally occurring cell protective mechanisms.

The biological foundation of the cell protective mechanisms is the presence of protective factors, including small molecules, cytokines, growth factors, and metabolic enzymes that can be activated in the event of injury. In the heart, for instance, numbers of protective factors have been identified and studied, including adenosine and bradykinin (small molecules); interleukin 6 (IL6) and cardiotrophin (cytokines); platelet-derived growth factors (PDGFs), fibroblast growth factors (FGFs), hepatocyte growth factor, and vascular endothelial growth factors (VEGFs) (growth factors); and superoxide dismutase (SOD) and catalase (metabolic enzymes). These factors and their protective actions are discussed here.

There are various cell protective factors released from the injured and activated cells during the different phases of an injury. During the early phase (within hours–days), adenosine and bradykinin can be released from injured cells [8–10]. Adenosine is a purine nucleoside, serving as the core structure of adenosine diphosphate (ADP) and adenosine triphosphate (ATP), which are involved in energy transfer and genome construction. Adenosine can also serve as a signaling molecule responsible for cell protection, suppression of inflammatory responses, relaxation of vascular smooth muscle cells, inhibition of the sinoatrial node activity [11], and downregulation of central nerve activities [12]. Bradykinin is a short peptide generated from its precursor kininogen by kallikrein-mediated cleavage. Bradykinin is well known for its roles in inducing vascular smooth muscle cell relaxation and protecting cells from injury [9]. These early protective factors can interact with cognate G protein-coupled receptors to activate intracellular signaling pathways involving phosphoinositide 3 kinase, protein kinase C (PKC), and mitogen-activated proteins kinases (MAPKs), supporting cell survival, mitigating cell injury, and prevent cell death [5,13]. These small molecules have been considered therapeutic agents for protection against ischemic myocardial injury.

Cytokines are a class of secreted proteins expressed in leukocytes, mast cells, vascular cells, epithelial cells, and fibroblasts [1,5]. Once expressed in cells, cytokines are released into the extracellular space to exert regulatory actions via interacting with cognate cell membrane receptors. Cytokines are involved in the regulation of physiological and pathological processes including inflammation, immune responses, hematopoiesis, cell proliferation, cell differentiation, cell death, and cell migration. Selected cytokines can participate in cell protective processes. Protective cytokines include, but are not limited to, IL6 [14], cardiotrophin 1 [15,16], IL8 [17], and Stromal cell-derived factor 1 [18]. These cytokines protect cells from injury and death by activating cell survival-related signaling pathways during the early period of injury (hours–days).

Growth factors are a large family of secreted proteins expressed in almost all nucleated cells. After expressed in the cell, growth factors are being released into the extracellular space to exert regulatory actions via interacting with cognate cell membrane receptors. Examples of growth factors include epidermal growth factors, FGFs, insulin-like growth factors, PDGFs, and VEGFs. Growth factors are responsible for the regulation of cell protection, survival, proliferation, differentiation, migration, and synthesis of the extracellular matrix. Their functions depend on the types and density of the growth factor receptors. Most growth factors can activate the protein tyrosine kinase (PTK) receptor signaling pathways to exert regulatory actions. The impact of a growth factor is dependent on its level in the extracellular space. An increase in the level of growth factor expression and release, usually in response to cell injury and death, can support cell survival and induce cell regeneration. Growth factors can act on the same cell types that produce the growth factors (autocrine signaling) and also on cell types in the neighborhood of the growth factor-producing cells (paracrine signaling) [1]. Growth factors are expressed and maintained at a low level under healthy conditions in adults to support cell survival and function. Their levels can be increased in response to various types of injury, induced by ischemia, microorganism infection, chemical toxicity, and mechanical impact. The purpose of growth factor upregulation in response to injury is to protect cells from death and facilitate wound healing by inducing cell regeneration [1].

A major class of metabolic enzymes involved in cell protection is bioenergetics-related enzymes, including superoxide dismutase and catalase. These enzymes can protect cells from reactive oxygen species (ROS)-induced injury. Common ROS molecules include superoxide and hydrogen peroxide, which are generated during electron transport through the mitochondrial respiratory chain for ATP production. ROS can react with and damage phospholipids, proteins, RNAs, and DNAs, causing cell injury and death. Superoxide dismutase can convert the highly toxic radical superoxide to less toxic hydrogen peroxide and can also oxidize superoxide to oxygen [19]. Catalase can break down hydrogen peroxide into water and oxygen [20]. Thus, these enzymes can convert toxic ROS to nontoxic molecules. To understand the mechanisms of these protective activities, it is helpful to review the processes of ROS generation during ATP production.

ATP is produced through the Krebs cycle and the electron transport processes in the mitochondrion, involving a series of bioenergetic enzymes. The Krebs cycle processes acetyl coenzyme A (CoA) derived from glycolysis and β oxidation of fatty acids to generate nicotinamide adenine dinucleotide (NADH) and succinate, which enter the mitochondrial electron transport chain for ATP production [21]. Glycolysis is a process that breaks down glucose to pyruvate. Each glucose molecule can generate two pyruvate molecules, which can enter the mitochondrial matrix [21] to form two acetyl CoA molecules through oxidative decarboxylation under the action of the pyruvate dehydrogenase complex [22]. Fatty acid β oxidation is a process that catalyzes fatty acids to acetyl CoA in the mitochondrial matrix [23]. All acetyl CoA molecules from glycolysis and fatty acid β oxidation can enter the Krebs cycle for generating NADH and succinate. Each Krebs cycle can generate three NADH molecules and one succinate molecule, all of which enter the mitochondrial electron transport chain for ATP production.

The mitochondrial electron transport chain consists of four enzyme complexes, referred to as complexes I through IV (CI–CIV), which are associated with the mitochondrial inner membrane, and also with two mobile electron carriers, namely ubiquinone (also known as Coenzyme Q (CoQ)) and cytochrome c, responsible for electron transport under the control of the enzyme complexes [24,25] (Fig. 1). Complexes I–IV function to oxidize and reduce substrates, including NADH, succinate, ubiquinone, cytochrome c, and oxygen, through which electrons are transported [24,25]. Among the electron transport complexes, complex I consists of NADH:ubiquinone oxidoreductase, responsible for transporting electrons from NADH to ubiquinone. Complex II contains succinate dehydrogenase that can oxidize succinate and transport electrons from succinate to ubiquinone via the mediation of flavin adenine dinucleotide redox reactions (FAD to FADH₂). Complex III comprises cytochrome bc₁ oxidoreductase responsible for transporting electrons from ubiquinone to cytochrome c. Complex IV consists of cytochrome c oxidase, which can transport electrons from cytochrome c to oxygen to form water [24,25].

The four enzyme complexes described above act in coordination to carry out the electron transport process (Fig. 1). This process begins with the oxidation of NADH (from the Krebs cycle) and reduction of ubiquinone (electron transport from NADH to ubiquinone) under the action of NADH:ubiquinone oxidoreductase (complex I). Succinate from the Krebs cycle is oxidized under the action of succinate dehydrogenase (complex II), and electrons from succinate are transported to FAD to form FADH₂, which in turn donates electrons to ubiquinone. The reduced form of ubiquinone from complexes I and II can be oxidized by cytochrome bc₁ oxidoreductase (complex III), which can also reduce cytochrome c (transporting electrons from ubiquinone to cytochrome c). The latter is then oxidized by cytochrome c oxidase (complex IV), an enzyme that can also fully reduce oxygen to water (transporting four electrons to O₂ to generate two H₂O molecules) [24,25]. The electron transport process is associated with proton release from the substrates of enzyme complexes I, III, and IV into the mitochondrial intermembrane space to generate a proton gradient from the mitochondrial intermembrane space to the mitochondrial matrix. This proton gradient creates energy that can be harvested by ATP synthase to phosphorylate ADP to ATP, a general form of energy for driving cell activities [24,25].

The ROS family members superoxide and hydrogen peroxide are generated through the electron transport processes at selected locations including complexes I, II, and III (primarily at complexes I and III), at which electrons leak from the enzyme complexes and the leaked electrons are accepted by oxygen molecules to generate superoxide [24–27] (Fig. 1). At these locations, superoxide is primarily released into the mitochondrial matrix. It can also be released into the mitochondrial intermembrane space at complex III. The generated superoxide can be converted to hydrogen peroxide under the action of SOD [25,28], and hydrogen peroxide is then turned into water and oxygen under the action of catalase [20]. Thus, the formation of superoxide can occur under healthy conditions and can increase when the demand for ATP rises under certain conditions such as excessive exercise [28,29] and activation of the epinephrine–β adrenergic receptor signaling system [30], which results in enhanced glucose metabolism and ATP production. The rate of superoxide formation can also increase under pathological conditions such as myocardial ischemia–reperfusion injury. Post-ischemic blood reperfusion brings oxygen rapidly into the ischemic myocardium, resulting in an increase in ATP production, a process associated with elevated superoxide formation. Superoxide-induced cardiomyocyte damage is the primary mechanism of reperfusion injury following a myocardial ischemic attack. In other forms of injury, which cause inevitably inflammatory responses, activated leukocytes can generate a much-increased level of superoxide [31,32]. Although superoxide is produced to eliminate microorganisms in the inflammatory site, it can also cause cell damage [31].

An injury can activate two forms of cell protection—short- and long-range protections. The short-range cell protective processes occur within the injured organ, whereas the long-range protective processes occur in distant organs that are not directly injured but are activated by messenger molecules from the injured organ—a mechanism recruiting all possible protective factors to minimize cell death [1]. These two types of cell protection are collectively defined as systems protective mechanisms (Fig. 2). For instance, in myocardial ischemia, both short- and long-range cell protective processes can be activated concurrently. The short-range cell protective processes occur within the myocardium. The long-range cell protective processes involve non-ischemic organs, such as the liver, bone marrow, and spleen [1]. The short-range cell protective actions include the release of paracrine small molecule protective factors such as adenosine [33,34] and bradykinin [35,36], and expression and release of cytokines [37,38] and growth factors [39–41]. These factors act to support cell survival and prevent cell death in the ischemic myocardium. In addition, metabolic enzymes, including superoxide dismutase and catalase, can be activated in response to myocardial injury to break down superoxide and hydrogen peroxide—reactive oxygen species that are elevated during the phase of blood reperfusion following coronary artery interventions (angioplasty and stenting) for the treatment of myocardial ischemia and can exacerbate cardiomyocyte injury [42–46]. All these shot-range activities protect the ischemic cardiomyocytes from death.

In myocardial ischemia, the long-range cell protective processes involve non-ischemic organs, such as the liver [47–49], bone marrow [50,51], and spleen [52,53]. Here, the liver is used to demonstrate this type of protection. The liver can be activated in response to messengers from the injured cells and activated leukocytes in the ischemic myocardium [47,49]. A potential messenger is the IL6 [9,10]. Other cytokines may also serve as messengers, but further investigations are needed to identify the cytokines involved. The activated liver can express and release endocrine cell protective proteins, including fibroblast growth factor 21 (FGF21) and trefoil factor 3 (TFF3) as detected by gene profiling [49], and can mobilize hepatic cells to the circulatory system [54]. The mobilized hepatic cells can go to the ischemic myocardium to locally deliver cell-protective factors [54] (Fig. 3). Taken together, the short-range and long-range cell protective mechanisms act in coordination and synergy to maximize the protective impact.

The presence of the naturally occurring cell protective mechanisms in myocardial ischemia is supported by evidence from numerous experimental tests, demonstrating that an induced mild “preconditioning” ischemic myocardial injury can effectively prevent myocardial infarction in a subsequent severe heart attack [3,5,6,55–57]. Myocardial preconditioning is indeed the most effective protective strategy against ischemic myocardial death to date [55–57]. Similarly, a mild “preconditioning” ischemic injury induced in a distant organ, such as the hind limb, can exert an effective cell protective impact in a subsequent heart attack [58–61]. It is now understood that a preconditioning injury can induce the expression and release of cell-protective factors from the ischemic heart and distant organs, which in turn prevent cardiomyocytes from death in subsequent myocardial ischemia [1]. It becomes clear that an important task in the research of cell protective biology is to identify, characterize, and understand the naturally occurring protective factors, which can be used as protective agents in the event of injury.

The protective actions are regulated by protective factors released from the injured and activated cells. In the case of ischemic myocardial injury, several types of short-range cell-protective factors have been studied, including adenosine, bradykinin, selected cytokines, and growth factors [1,2,5]. Adenosine exerts a cell protective effect by interacting with G protein-coupled receptors, including adenosine receptors A1, A2A, A2B, and A3 [12]. The adenosine A1 and A3 receptors are associated with inhibitory Gi proteins that suppress the activity of adenylate cyclase (an enzyme catalyzing the formation of cAMP from ATP), reducing the formation of cAMP. The A2A and A2B receptors can interact with stimulatory Gs proteins that activate adenylate cyclase, inducing cAMP formation. The A2B and A3 receptors are also associated with Gq proteins, which can activate the phospholipase Cβ signaling pathway, increasing the cytosol calcium level and enhancing the activity of PKC and MAPKs [5]. These enzymes support cell survival and stimulate cell regeneration.

Bradykinin exerts cell protective actions by binding to G protein-coupled receptors [1]. Two bradykinin receptors have been identified: bradykinin receptors B1 and B2. Both receptors can activate Gq proteins and the associated phospholipase Cβ signaling cascade, resulting in the activation of the mitogen-activated protein kinase signaling pathway, which supports cell survival. In myocardial ischemia, bradykinin can be rapidly released from injured cells to participate in cell protective actions by binding primarily to the bradykinin B2 receptor. Bradykinin and its agonists have been considered potential pharmacological agents for protection against ischemic myocardial injury.

In myocardial ischemia, selected cytokines, including IL6 and cardiotrophin 1, can activate the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling pathways to support cardiomyocyte survival [1,2,58]. IL6 can bind to IL6 receptor α (IL6Rα), causing recruitment of two gp130 molecules to the IL6 receptor. The two gp130 molecules can recruit JAKs, one to each gp130 molecule. This action triggers autophosphorylation (reciprocal phosphorylation) of the two JAK molecules, which in turn phosphorylate the two gp130 molecules. Each phosphorylated gp130 molecule can recruit a STAT molecule, which is phosphorylated by JAK. The two phosphorylated STAT molecules on the two gp130 molecules then dissociate from the IL6 receptor/gp130 complex and form a dimeric STAT complex. This complex serves as a transcriptional factor capable of activating genes responsible for inflammatory, cell-protective, and cell-regenerative actions [1]. Cardiotrophin 1 can activate a similar signaling pathway, causing similar actions.

Growth factors, such as PDGFs, can stimulate the PTK receptor—MAPK signaling pathways to support cell survival [1,2,5,62]. PDGFs are a growth factor family composed of PDGF A, PDGF B, PDGF C, and PDGF D. These proteins are present in the dimeric form in the extracellular space. The most abundant PDGF dimers include PDGF AA, AB, and BB [1]. PDGFs can bind to PDGF receptors (PDGFRs), including PDGFRα and PDGFRβ. PDGF A and PDGF C can bind to PDGFRα; PDGF B to both PDGFRα and PDGFRβ; and PDGF D to PDGFRβ. The binding of a PDGF dimer to a receptor can trigger dimerization of two PDGFRs. This action causes tyrosine autophosphorylation of the two PDGF receptors and recruitment of the growth factor receptor-bound protein 2 (Grb2)/son of sevenless (SOS) complex. The Grb2/SOS complex can then activate a signaling cascade consisting of Ras, mitogen-activated kinase kinase kinases, MAPKKs, and MAPKs. MAPKs can in turn phosphorylate Fos and Jun proteins. Phosphorylated Fos and Jun can form a complex known as activating protein 1 (AP1). All phosphorylated Fos, Jun, and AP1 can serve as transcriptional factors to cause the expression of genes responsible for regulating cell survival, proliferation, and differentiation [1].

In addition to the short-range cell-protective factors discussed above, the liver can express and release endocrine protective factors in response to ischemic myocardial injury, including FGF21 and TFF3 [47]. FGF21 is one of the 22 FGF family members, was originally identified in the pancreas, and has been known to lower the level of blood glucose and prevent obesity [63–66]. In the ischemic myocardium, FGF21 can bind to FGF receptor 1 in cardiomyocytes to activate the phosphoinositide 3-kinase–phosphatidylinositol 3,4,5-trisphosphate–Akt serine/threonine kinase 1 pathway to protect cardiomyocytes from death [49].

TFF3 is one of the three trefoil factor family members originally found in the intestinal epithelial cells and plays a role in protecting the intestinal epithelium [67,68] and brain [69] from injury. In the case of myocardial ischemia, TFF3 can alleviate cardiomyocyte injury via its inhibitory effect on intra-cardiomyocyte calcification, a process causing cardiomyocyte death. Cardiomyocyte calcification occurs within ∼30 min following the induction of experimental myocardial ischemia, a result of annexin translocation from the cell membrane to actin filaments. Annexins are a family of intracellular Ca²⁺-binding proteins that anchor to the inner surface of the cell membrane via the mediation of Ca²⁺ [70–75]. These proteins can be freed when the cell membrane degrades in myocardial ischemia. Free annexins can bind to actin [71], a property promoting the accumulation of annexins on the actin filaments of cardiomyocytes. The Ca²⁺ ions on the surface of free annexins can be densified to attract phosphate ions for the formation of calcium phosphates. Thus, annexin accumulation on actin filaments can facilitate calcium phosphate aggregation to cause cardiomyocyte calcification. The liver-derived endocrine factor TFF3 can bind to the site of annexin responsible for interacting with actin, thereby blocking annexin binding to the actin filaments of cardiomyocytes and preventing annexin and calcium phosphate accumulation (Fig. 4). Thus, TFF3 represents a potential anti-cardiomyocyte calcification factor that can prevent cardiomyocyte injury.

Myocardial ischemia can cause hepatic cell mobilization to the ischemic myocardium to locally release cell-protective factors, including FGF21 and TFF3, a seemingly more efficient way to deliver protective factors than endocrine delivery from the liver to the heart [1,2,54]. The regulation of hepatic cell mobilization in myocardial ischemia involves several processes. Injured cells and activated leukocytes in the ischemic myocardium can release cytokines, which can activate circulatory leukocytes [76]. The activated leukocytes can infiltrate the hepatic parenchyma and express matrix metalloproteinase 2, which can in turn degrade the collagen matrix to cause hepatic cell mobilization into the circulatory system [76]. The ischemic myocardium, but not the intact myocardium and other organs, can retain the mobilized hepatic cells, which subsequently release cell-protective factors to the ischemic myocardium [54].

Cell protective engineering is to develop engineering strategies and technologies to optimize protective actions against cell death in injury and disease. Cell protective engineering strategies can be developed based on naturally occurring protective mechanisms. The rationale for cell protective engineering is that the naturally occurring protective actions are not controlled to the optimal level in the timing of actions and the level of effectiveness—not all cell-protective factors can be released to reach the optimal levels prior to cell death following an injury. For instance, in myocardial ischemia, the protective growth factor genes can only be expressed and released after ∼12 h, whereas cardiomyocyte death starts within ∼1 h in the core region of myocardial ischemia [49]. Thus, it is necessary to develop and utilize cell protective engineering strategies to optimize the timing and level of protective actions.

Cell protective engineering strategies can be established at the molecular, cellular, and tissue levels. At the molecular level, cell protective factors, such as the small molecules adenosine and bradykinin and the proteins VEGF, FGF21, and TFF3, can be prepared and administered individually or in various combinations into the injury site or the circulatory system promptly. A sustained cell protective effect can be achieved by transferring protective gene(s), such as the FGF21 and/or TFF3 genes, into injured cells [1]. As it takes time to express the transferred genes, it is necessary to co-deliver cell-protective proteins and genes to cover the entire period of cell death (∼30 min to ∼10 days following experimental myocardial ischemia [47]). Although cell-protective factors are often used individually in experimental and clinical tests, a combination of multiple protective factors has been proven more effective in experimental myocardial ischemia [47]. In addition, the administration of superoxide dismutase can reduce the level of myocardial ischemia–reperfusion injury [77,78]. Further investigations are needed to identify more naturally occurring cell-protective factors and evaluate the effectiveness of these factors for clinical applications.

At the cellular level, protective cells, which can express and release protective factors, can be prepared and transplanted to the injury site for regional delivery of protective factors. For instance, bone marrow-derived hematopoietic stem cells have been utilized for transplantation into the ischemic myocardium to release protective factors, an effective approach to alleviate myocardial infarction [79,80]. A similar cell protective effect has been observed by transplanting hepatic cells into the ischemic myocardium [54]. Overall, any cells that express cell protective genes can be potentially utilized for this purpose.

At the tissue level, biomaterial scaffolds can be constructed by using collagen matrix or synthetic polymers, impregnated with cell protective proteins and/or genes, and/or seeded with protective cells [1,81]. Such scaffolds can be applied to injured organs for cell protection. For instance, in myocardial ischemia, a sheet-like scaffold can be placed and secured onto the surface of the ischemic myocardium to protect the ischemic myocardium from rupture and deliver cell-protective proteins and/or genes into the ischemic myocardium to alleviate cardiomyocyte death [1]. Taken together, the molecular, cellular, and tissue-level protective engineering strategies can be developed and utilized toward optimal control of the types, the timing of action, levels, and coordination of cell-protective factors to maximize the cell protective impact.

Cells can naturally protect themselves from death in the event of injury; however, the capacity of cell protection is limited because of inadequate timing and effectiveness of the protective actions. These problems can be addressed by developing and applying protective engineering strategies to injured cells to better control the timing and level of protective actions, thereby reducing the rate of cell death. Although the impact of cell protective engineering research has been demonstrated, the protective engineering strategies remain to be improved. Major obstacles include the lack of a complete list of cell-protective factors involved in each form of injury and the lack of understanding of the synergistic actions of various regional and distant protective factors. Overcoming these obstacles is the primary task of research in cell protective engineering.

• US National Science Foundation, Grant No. 1403036.

The author declares no conflicts of interest.

The datasets generated and supporting the findings of this article are obtainable from the corresponding author upon reasonable request.