As stated above, different domains of the TSP can interact with corresponding receptors, including integrins, CD36 and CD47. Integrins are a class of glycosylated, heterodimeric transmembrane receptors that consist of α and β subunits. The interaction of cardiac ECM with specific cell surface integrins is the foundation of cardiomyocyte maturation and remodeling (16).

Moreover, the interaction between TSPs and integrins participates in vascular remodeling and pathology. TSP-1 and TSP-2 are regarded as potent endogenous antiangiogenic proteins that directly affect CD36, CD47, and integrins. Vascular cells express various integrins that bind to TSP-1, including α_Iibβ₃, α_vβ₃, and α₅β₁ (17). The N-termini of TSP-1 and TSP-2 can be recognized by α₃β₁, α₄β₁, and α₆β₁ to mediate the adhesion of vascular ECs (18). The binding of integrin α₃β₁ to TSP-1 promotes EC migration, cell motility, and antiangiogenic effects (19). In another study, TSP-1 was shown to bind to αvβ1 and regulate the nuclear shuttling of Yes-associated protein in response to mechanical stress-induced vascular injury, leading to dynamic remodeling of the aorta in mice (20).

The TGF-β superfamily has a significant role in inducing tissue fibrosis and inflammation. Type I repeats of TSP can interact with latent TGF-β. For instance, TGF-β signaling enables the ERK1/2 pathway to activate fibroblasts and induce interstitial fibrosis in the aging course (21). The TGF-β family is mainly classified into the TGF-β1, -2, and -3 subfamilies. One of the downstream mediators of TGF-β1, connective tissue growth factor (CTGF), also plays an important role in fibrosis.

TSP-1 is a major mediator of TGF-β activation, increasing CTGF and collagen levels and the accumulation of extracellular matrix proteins (22). All of these factors stimulate and are hallmarks of tissue fibrosis. Increased TSP-1 in intermittent hypoxia (IH) patients and mice, which activates the TGF-β pathway via JAK2/STAT3/TSP-1 signaling, has a significant effect on IH-induced fibroblast activation and cardiac fibrosis (23). A TSP-1 antagonist that blocks TGF-β activation can reverse myocardial fibrosis and ensure left ventricular function in hypertensive diabetic rats (24). TSP-1 acted as a protective factor by maintaining fibroblast function and matrix metabolism in pressure-overloaded myocardium. TSP-1 is also downstream of the TGF-β signaling pathway and has been identified as a regulator of microtube formation in glioblastoma (25). In addition to the heart, as a result of TSP-1/TGF-β pathway upregulation, collagen deposition and extensive fibrosis in the arterial wall led to arteriosclerosis, while in TSP-1 knockout (KO) mice or mice treated with TSP-1 antagonist, arterial collagen, CTGF, and arterial stiffness were decreased (26). Ying Xia et al. reported that deletion of TSP-1 disrupted TGF-β signaling, leading to impaired myofibroblast differentiation and decreased collagen expression (27).

TSP-2 can bind to latent TGF-β but cannot activate the TGF-β signal and is regarded as a competitive binding factor to TSP-1 in CVD. However, in the cancer microenvironment, a high level of TSP-2 produced by cancer-associated fibroblasts is activated via the TGF-β1/Smad2/3 pathway, binds to integrin α_vβ₃/CD36 and activates the MAPK pathway in cancer cells to promote tumor growth and adhesion (28).

Regarding injury to other organs, TSP-1 is highly expressed in patients with sepsis-induced acute kidney injury (AKI). The transcription factor USF2 activates TSP-1 to activate the TGF-β/NLRP3/Caspase-1 signaling pathway, resulting in promotion of the oxidative stress response and stimulation of pyroptosis in sepsis-induced AKI (29). TSP-1 deficiency protects mice against sepsis-induced AKI by decreasing the expression of inflammatory and apoptosis-promoting cytokines, such as the NLRP3 inflammasome, caspase-1, IL-1β, and IL-18, which increase cell viability and partially reverse cell pyroptosis. TSP-1 deletion reduces TGF-β signaling and protects against renal fibrosis in a high-fat diet mouse model (30). In another study, interstitial macrophages secreted TSP-1 after hypoxia exposure, and pathological TSP-1 promoted TGF-β activation and Rho-kinase-mediated vasoconstriction in mice, resulting in pulmonary hypertension (31).

The endoplasmic reticulum (ER) is a large protein processing and transporting region. Mistaken ER protein folding leads to the accumulation of unfolded and misfolded proteins. This disturbance of ER homeostasis initiates the protective stress response and is known as the unfolded protein response (UPR) or ER stress (32). Protein kinase R-like ER kinase (PERK), activating transcription factor 6α (ATF6α), and inositol requiring enzyme 1 alpha (IRE1α) are three primary sensors that strengthen protein folding under ER stress (33). ER stress is a double-edged sword that can restore cell homeostasis and may lead to cell defects (32). In TSP-1-overexpressing transgenic mice, TSP-1 binds to PERK and induces the downstream factor ATF4, thus activating ER stress and inducing autophagy-mediated lethal cardiac atrophy (34).

However, for the other member of group A, TSP-2, further research is needed to reveal the possible links between TSP-2 and ER stress.

Nitric oxide (NO) is a vital physiological regulator of vasomotion and blood flow. TSP-1 affects the proliferation, migration, and angiogenesis of SMCs, ECs, and platelets (PLTs) by regulating NO signaling (22, 35). TSP-1 binds to the cell surface proteins CD47 and CD36 and subsequently participates in the activity of endothelial nitric oxide synthase (eNOS) (36, 37). Moreover, the combination of TSP-1 with CD47 or CD36 can inhibit the SMC cyclic guanosine monophosphate pathway in the presence of a low dose of NO (NO/cGMP) (38), exerting antiangiogenic effects and activating apoptosis in microvascular ECs, which leads to failed endothelial tubule formation and directly affects other antiangiogenic activities. Studies have shown that, compared with CD36, CD47 has a greater affinity for TSP-1 and inhibits NO signaling (39). Through binding to CD47, TSP-1 inhibits eNOS activation and arterial relaxation, manifesting as a blood pressure booster agent (40).

TSP-1 engages the receptor CD47 of ECs to mediate cell phenotypic transformation, and the activation of CD47 inhibits the bioavailability of VEGF (41, 42), as observed in lymphatic endothelial cells, resulting in AKT-eNOS signaling activation and NO reduction (43). Thus, silencing CD47 blocks lymphangiogenesis and atherosclerotic lesion formation (43). Deletion of TSP-1 protects against inflammatory lesion development and vascular smooth muscle cell (VSMC) phenotypic transition in leptin-induced atherogenesis (44). TSP-1 can also suppress the NO signaling pathway by interacting with integrin to regulate platelet aggregation (39).

TSP-2 seems to be an NO target, and suppressing TSP-2 relieves the eNOS-knockout phenotype without restoring NO signaling in mice with injury- and ischemia-induced angiogenesis (45).

Matrix metalloproteinases, such as matrix metallopeptidase (MMP)-2, MMP-3, and MMP-9, participate in tissue remodeling by binding to TSP type I repeats (27).

TSP-1 deficiency increases myocardial MMP-3 and MMP-9 activation under pressure overload, resulting in early cardiac hypertrophy and late dilation. During remodeling of the diabetic heart, TSP-1 deletion increases MMP-2 and MMP-9 activation to degrade collagen and inhibit fibroblast function (46).

TSP-2 acts as a key regulator of cardiac matrix integrity, is required for the myocardium to respond to pressure overload, and plays a role in regulating MMP activity (47). TSP-2 plays a protective role in age-related cardiomyopathy by activating the Src/Akt survival pathway, decreasing inflammation and MMP-2 activity, and maintaining collagen crosslinking (48). High levels of MMP-2 (49) and MMP-9 (50) may cause local disruption of the myocardial matrix, leading to cardiac rupture and dilatation after Ang II infusion in TSP-2-deficient mice (47). Enhanced matrix destruction is shown in the heart of doxorubicin (DOX)-induced TSP-2 KO mice and is accompanied by increased levels of MMP-2 (51). A lack of TSP-2 increases the levels of MMPs in the extracellular matrix and helps with the degradation of fibrillar collagen, thus reducing fibrosis around cardiac cell grafts (52).

MMPs also take part in vascular physiology. TSP-1 can be hydrolyzed into proteolytic fragments, and the different fragments exert pro- or antiangiogenic effects by activating the MAPK pathway to mediate the MMP/tissue inhibitor of metalloproteinase (TIMP) balance (53). TSP-2 inhibits angiogenesis by regulating EC function and modulating MMP-2 and MMP-9 (54).

Reactive oxygen species (ROS) are associated with senility and injury, regulating myocardial metabolism and contributing to vascular dystonia (55). The family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isozymes is the main source of ROS. Nox-4-based NADPH oxidases are the main source of ROS in the vasculature, and downregulation of Nox4 in SMCs inhibits neointimal hyperplasia by decreasing TSP-1 and suppressing SMC proliferation (56). Hypoxia-responsive TSP-1 mediates the critical event in pulmonary hypertension. The level of TSP-1 was increased in hypoxia-induced human pulmonary artery SMCs, and TSP-1-stimulated Nox4 expression was enhanced, causing SMC proliferation and high blood pressure (57). O₂⁻ is the first product induced by Nox and rapidly changes into stable H₂O₂. Moreover, excess accumulation of O₂⁻ disrupts the balance of O₂⁻/H₂O₂, resulting in the impairment of coronary arteriolar vasodilation and heart ischemia (58, 59).

In diabetic conditions, TSP-1 binds to the cell-surface receptor CD47 and significantly increases Nox1-mediated ROS and O₂⁻ production, causing endothelial senescence and vascular dysfunction (60–62), while TSP-2 plays an antiangiogenic role. Increasing NADPH oxidase activity and oxidative stress induce the production of TSP-2, which induces bone marrow-derived angiogenic cell dysfunction and vascular impairment (63). The TSP-1/CD47 axis also activates signal-regulatory protein-α, another kind of cell-surface receptor of inflammatory cells, increasing O₂⁻ production and promoting renal ischemia‒reperfusion injury (64). H₂O₂ can induce vasodilation through the release of prostaglandin E2 and calcium-activated potassium channel-related SMC hyperpolarization.

Activation of the plasmin/plasminogen system is necessary for regulating angiogenesis in a variety of diseases (65). A study indicated that plasminogen decreases the expression of TSP-1 and TSP-2, thereby enhancing angiogenesis in damaged brain tissue to help cells resume after ischemic stroke (66). However, no additional studies have evaluated the relationship between TSP-2 and the plasmin/plasminogen system.

The absence of plasminogen suppresses EC migration and decreases cerebrovascular density. Platelet activation releases stored TSP-1 in platelet α-granules; thus, plasma TSP-1 is significantly increased during the acute stage in ST-segment elevation myocardial infarction (STEMI) patients and decreases 1–3 days and 3 months after percutaneous coronary intervention (PCI), which might be associated with major adverse cardiac events (67).

In addition, both plasmin and TSP-1 can activate TGF-β1 signaling in the fibrotic process (68, 69). However, TGF-β1 activation is bistable: the plasmin-mediated mode is characterized by low activation, and the TSP-1-mediated mode is characterized by high activation. Interestingly, when both plasmin and TSP-1 are present, increasing plasmin can disrupt the TSP-1/TGF-β1 feedback loop and thus cause proteolytic cleavage of TSP-1 and inactivation of TGF-β1 signaling (70). This phenomenon corroborates that the activation of plasmin inhibits the expression of TSP-1.

Activation of the TSP-1/CD47 pathway plays a crucial role in the excitation and migration of T regulatory cells (Tregs) to inhibit inflammation in atherosclerosis and abdominal aortic aneurysm (AAA) (71, 72). TSP-1 and TSP-2 also interact with CD47 on the surface of T cells, which triggers T-cell apoptosis to block inflammation. In CD47-knockout mice, TSP-1- or TSP-2-knockout mice, prolonged inflammation occurs along with defects in T-cell apoptosis (73). TSP-2 is also an important regulator of T-lymphocyte migration and differentiation through its interaction with CD47 (71) in humans and mice (74). TSP-2 deficiency results in the generation of fewer Tregs and lower IL-10 and IL-10 receptor levels, thereby leading to an imbalance in the immune response and cardiac damage in Coxsackievirus group B type 3 (CVB3) virus-induced myocarditis (74, 75).

MMPs are necessary for macrophages to persist and migrate into the ECM to induce an inflammatory response in AAA (76). TSP-1 deficiency upregulates the tissue inhibitor MMP-1, which suppresses extracellular gelatinase activity and inhibits MMP activation, thus alleviating AAA development (77). TSP-1 deficiency also enhances atherosclerotic plaque maturation by accelerating inflammation, and macrophage-induced MMP-9 contributes to elastin degradation (78). TSP-1 can bind to CD47 in macrophages to enhance the inflammasome-dependent maturation of IL-1β, which promotes inflammation in response to lipopolysaccharide (LPS) (79). TSP-1 may promote proinflammatory macrophage differentiation to aggravate human aortic dissection (AD) (80). Another study showed that the overexpression of TSP-2 promotes the polarization of macrophages toward an anti-inflammatory phenotype by activating the PI3K pathway in vitro and attenuating LPS-induced pulmonary inflammation (70).

TSP-1 serves as a master regulator of cancer invasion (81, 82). A study showed that the expression of TSP-1 and CD47 is increased in human malignant melanoma tumor tissue, and targeting the TSP-1/CD47 pathway may preserve CD8⁺ T-cell activation, proliferation, and bioenergetics to alleviate the tumor burden (83). By the way, downregulating TSP-1 levels in skin dendritic cells effectively promoted antitumor reactions through increasing tumor-infiltrating CD4⁺ and CD8⁺ T cells. Moreover, TSP-1-knockout bone marrow-derived DCs retarded tumor growth, while targeting TSP-2 did not have the same effect (84). This finding was opposite to the antitumor angiogenesis response of TSP-1/2, indicating that systemic antitumor treatment with TSP-1 could be a double-edged sword.

Group B TSPs binds to integrin to regulate physiological and pathological processes. Overexpression of TSP-3 in the mouse heart significantly damages cardiomyocyte integrity due to reduced sarcolemmal residence of integrins, such as α₇β_1D and β₁ (85). Studies have shown that the expression of TSP-5 is decreased in the hearts of DCM patients and that a lack of TSP-5 in mice spontaneously results in DCM at a young age (86). TSP-5 interacts with cardiomyocyte integrin β₁ to maintain cardiomyocyte homeostasis, and TSP-5 or integrin β₁ deficiency results in similar F-actin dissolution, connexin-43 defects, and spontaneous apoptosis (86).

The interaction of TSP-4 and α_Mβ₂ and β₃ integrins in macrophages activates p38-MAPK signaling (87). The p38-MAPK pathway plays a pivotal role in the production of proinflammatory mediators and cytokines, as well as in endothelial-leukocyte interactions, resulting in vascular inflammation and arteriosclerosis (87–89). TSP-4 also modulates the proliferation of SMCs and ECs, thereby exerting atherogenic effects (90). Thus, TSP-4 serves as a proangiogenic factor in wound healing. TSP-4 regulates the adhesion, migration, and proliferation of EC cells by interacting with integrin α₂ and gabapentin receptor α₂δ-1 (91), resulting in a strong proangiogenic effect in vivo and in vitro.

Bone marrow-derived TSP-5 mediates atherosclerotic calcification, and a lack of TSP-5 induces the atherogenic and osteogenic phenotype in macrophages via integrin β₃ (92). TSP-5 is involved in the chemotaxis and attachment of VSMCs and inhibits osteochondrogenic phenotypic switching in VSMCs, thereby inhibiting vascular calcification (93, 94). Increased TSP-5 levels during injury or other pathological processes exert protective effects by maintaining the contractile phenotype of VSMCs through interactions with the integrin α₇β₁ (93, 94). In addition, the C-terminus of TSP-5 binds directly to integrin α₅, blocking aberrant activation of ECs in mice and hence reducing vascular inflammation and atherosclerosis (95).

TSP-4 and TSP-5 regulate atherosclerosis, aortic aneurysm, and other vascular tissue remodeling processes through the TGF-β signal pathway (96). Activated TGF-β1 stimulates the Smad3 pathway to upregulate endothelial TSP-4, leading to EC adhesion, migration, and proliferation (97). This strong proangiogenic function can be blocked in TSP-4 KO mice. Smad3 activation in ECs also upregulates TSP-4 expression and angiogenesis via TGF-β during tumor growth (97).

TSP-5, also known as cartilage oligomeric matrix protein (COMP), participates in the differentiation of stem cells and passaged chondrocytes and is dependent on TGF-β signaling (98). TGF-β1 binds to the C-terminal domain of TSP-5, and an additional binding site is the 3-repeat TSP in the presence of manganese. This combination increases TGF-β1-dependent transcription and enhances its bioactivity (98). Along with the activation of the TGF-β signal, the levels of TSP-5 are also elevated, which plays a critical role in skin fibrosis by promoting collagen deposition and modifying fibroblast functions (99, 100). Bone morphogenetic protein (BMP)-2 is a member of the TGF-β superfamily. Yaoyao Du et al. suggested that TSP-5 binds to BMP-2 via the C-terminus to prevent its interaction with the BMP-2 receptor, inhibit the osteochondrogenic transition of VSMCs, and improve vascular calcification (101).

All three members of group B can active ER stress to regulate tissue development. TSP-4 is expressed in the heart and skeletal muscle during injury and damage, and it contributes to sarcolemmal stability and cardioprotection through binding to ATF6α-activated ER stress (102–104). TSP-4 is prominently localized within intracellular vesicles and the ER or its compartments in myocardial cells, with minor accumulation in the extracellular space (102). As a result of ER stress, TSP-4 KO mice infused with Ang II have exaggerated hypertrophic hearts and a high incidence of aneurysm but a protective role in endothelium-dependent relaxation in resistant arteries (105). TSP-3 is highly similar in structure to TSP-4 and is upregulated during cardiac disease. Although TSP-3 also activates ER stress by binding to ATF6α, TSP-3 oppositely enhances cardiac pathological conditions by inhibiting intracellular integrin and destroying myocardial membrane stability (85). The familial mutation in type 3 repeats of TSP-5 induces pseudoachondroplasia and multiple epiphyseal dysplasia resulting from pathological accumulation of mutated TSP-5 in the rough ER and apoptosis of the cells (106). Thus, mutated TSP-5 induces ER stress to regulate cartilage development.

TSP-4 promotes endothelial dysfunction and contributes to the process of hypertension by impairing NO bioavailability and blocking vascular vasodilation in Ang II-infused mice (105). TSP-5 plays a protective role in BP control by improving endothelium-dependent relaxation through CaKMII/eNO signaling. It binds to the C-terminus of Piezo1, which promotes intracellular Ca²⁺ influx, eNOS activation, and NO generation resulting from activation of endogenous Piezo1 currents (107). TSP-3 has not been found to regulate CVDs through NO signaling.

It is unknown whether TSP-3 acts on immune cells to affect the cardiovascular system. TSP-4 expression directly supports macrophage functions and switching of the proinflammatory phenotype during atherogenesis (108). Inflammation stimulates the expression of TSP-4 in macrophages, while increased TSP-4 promotes the accumulation and proinflammatory phenotypic differentiation of macrophages in LPS-induced peritonitis (108). In vascular inflammation, recombinant TSP-4 contributes to injury-induced restenosis by accelerating macrophage adhesion to VSMCs and increasing VSMC proliferation and migration (109). TSP-5 is also critical for inducing the beneficial phenotype of VSMCs and macrophages to help maintain vascular integrity and function (110, 111). The deletion of TSP-5 promotes VSMC migration and exacerbates VSMC calcification and atherosclerosis (111); moreover, this deletion results in a phenotypic shift of macrophages to atherogenic and osteogenic characteristics (112).