RAAS and TGF-β are the canonical regulators in vascular fibrosis. Although they are not new to public attention, it is still necessary to be elaborated for the better understanding of newfound evidences. Initially, studies on RAAS concentrated on hypertension and heart failure, but later shifted to vascular fibrosis. TGF-β has been identified as an essential mediator in fibrotic parenchymal organs. In the case of vascular fibrosis, however, a linkage between RAAS and TGF-β is revealed.

According to research findings, mitochondrial oxidative stress is increased in cardiac remodeling and failure (107), and is highly linked to inflammation and fibrosis (41). It is well acknowledged that OS is characterized by an overabundance of ROS, which can be generated by aberrant mitochondrial metabolism and lead to mitochondrial malfunction. Mitochondrial DNA is extremely vulnerable since oxidative damage may set off a vicious cycle of ROS generation (42). Ang II has also been demonstrated to promote mitochondrial dysfunction in vascular endothelial cells, through increasing ROS production (43). Besides, mitochondrial dysfunction interacts with TGF-β on various levels to promote or inhibit tissue fibrosis (108). Histological and western-blotting studies show that high-fat diet (HFD) animals had an increase in cardiac and aortic fibrosis biomarker genes such as Col-1/3, CTGF, and TGF-β, as well as aortic media and carotid intima media thickness (44, 109). It is noteworthy that aforementioned abnormalities can be reversed by MitoQ, a mitochondrial antioxidant (44, 45), which partly contributes to the of OS induces fibrosis.

On the other hand, ER stress is associated to mitochondrial OS. ER stress protein markers, BiP and PDIA6, was also found increased in HFD mice and positively correlated with cardiac interstitial fibrosis (44). Luckily, MitoQ can relieve those variations. Further experiments on fibroblasts in vitro suggest the same, for that MitoQ and ER stress inhibitor (4-phenyl butyric acid) alleviates Ang II induced fibrotic alternations (44). Besides, research on human umbilical vein endothelial cells have also shown that Aldo suppresses the expression of SOD2 protein, mitochondria DNA and protein through MR. Intriguingly, another mitochondrial antioxidant (Mito-TEMPO) can also twist this attenuation (46). These evidences further indicate OS is taking part in fibrosis in an ER stress-dependent manner, and mitochondrial OS antagonists like MitoQ and Mito-TEMPO possess their therapeutic benefits. Besides, scholars discovered that particulate matter 2.5 (PM2.5) exposure triggers aortic fibrosis via mitochondrial OS with the mechanisms mentioned above (45).

Mitochondria serve as the cell's energy factory, in which fatty acid binding protein 3 (FABP3) subtly modulates the process of fatty acid oxidation (FAO) (110). FAO generates ATPs as an energy source during fibrosis, for VSMC proliferation, ECM protein secretion, and immune cell functions (110). Interestingly, recent research reveals that FABP3 elevation induces aortic adventitia fibroblasts growth and ECM deposition (47), hence contributing to vascular fibrosis. Immunohistochemistry data demonstrate a substantial relationship between the expression of FABP3 and adventitia ECM proteins, including Col-1, FN-1, MMP-3/9 (47). However, another research on FABP3 suggests the opposite. FABP3^−/− mice exhibited severer cardiac hypertrophy than wildtype mice owing to FAO dysfunction, which enhances glycolysis and toxic lipid accumulation (48). As a conclusion, neither elevation nor deficiency in FABP3 is beneficial, meaning that mitochondrial energy metabolism has deeper mechanism that merits further investigation.

Last but not least, emerging studies have corroborated that mitophagy effects mitochondrial dysfunction in organs and tissues fibrosing (111). Consequently, there emerges plenty of potential therapeutic targets, such as targeting mitophagy, miR-30a, mTORC1/PGC-1 axis, and targeting mitochondrial quality control (108, 112, 113). These targets have the potential to improve mitochondrial function and reduce oxidative stress, thereby slowing or reversing the accumulation of ECM. Besides, employing wide tactics of genetic engineering, small compounds targeting mitochondria, and mito-therapy are all therapeutic options.

EndMT, as mentioned above, is pathological condition of endothelium that results in vascular fibrosis. It occurs when ECs cease to secrete their particular biomarkers, but instead products of mesenchymal and myofibroblasts like Col-1 and α-SMA (49). The problem is that EndMT not only manifests a dysfunction of endothelium integrity and altering to a migratory form, but also turns ECs into invasive and proliferative phenotype, all of which are contributive to fibrosing. Multiple signaling pathways are involved in the formation of EndMT, for instance, IL-1β, IL-6 and TNF-α can be triggers of TGF-β pathways via NF-κB (50). Meanwhile, IFN-γ acting through JAK/STAT pathway and ET-1 participation in TGF-β/Smad signaling as well promotes EndMT process (51, 52). Unfortunately, EndMT causes certain dysfunctions in vascular system. For instance, NO production in ECs was essential to maintaining integrative structures and functions of vessels, whereas growing EndMT causes NO shortage. Besides, EndMT also leads to vascular fibrosis related ROS accumulation, vascular inflammation and ECM deposition. Furthermore, evidence manifests that ischemia-reperfusion injury in particular, can be the direct trigger of EndMT, which in turn promotes atherosclerosis and arteriosclerosis, especially in ischemic stroke in brain (53). However, a circulating human microRNA family named let-7 has been identified to play crucial role in remodeling the blood-brain barrier and reversing EndMT caused by ischemic stroke via a TGF-βR1/let-7i double-negative feedback pathway (53).

Recently, hydrogen sulfide (H₂S), an essential mediator, generates from L-cysteine through enzymatic reactions, has been established as a blocker of EndMT and vascular fibrosis process. There are currently four major approaches. Initially, H₂S acts as a vasodilator by inhibiting the TGF-β/cGMP-PKG pathway and lowering RAAS (54, 114), especially in chronic hypertension-induced vascular alteration. Secondly, H₂S can boost endothelial nitric oxide synthase (eNOS) for more sufficient NO synthesizing in guarding against EC dysfunctions and reducing blood pressure (55). Additionally, H₂S also has an anti-inflammatory impact by blocking the migration of CD11b⁺ leukocytes (56). Finally, H₂S has been demonstrated to be a negative regulator of OS, which is thought to be connected to EndMT (57). The foregoing facts have surely pushed H₂S's function into the future treatments for vascular fibrosis.

Inflammation is a key factor in the development and progression of fibrosis. Inflammatory cells and mediators are playing a crucial role in myofibroblast activation and ECM deposition. Understanding the cellular and molecular mechanisms of fibrosis may lead to the development of new therapeutic interventions.

We have so far elaborated the molecular mechanisms of vascular fibrosis from several different aspects including RAAS and TGF-β, mitochondria, EndMT, as well as inflammation. However, there are several more factors closely related to vascular fibrosis that cannot be overlooked.

Marinobufagenin (MBG), an endogenous cardiotonic steroid being identified in mammalian extracellular fluids, promotes vascular fibrosis by inhibiting friend leukemia virus integration 1 (Fli-1) along with activating TGF-β1/Smad pathway (81). Since Fli-1 function as a nuclear inhibitory transcription factor to Col-1 gene, MBG elevation results in Col-1 overexpression (82). Interestingly, spironolactone has shown alleviation to the profibrotic effect of MBG (83). ROCK (Rho-associated coiled-coil forming protein kinases), which expresses in most vascular cells (ECs, VSMCs, fibroblasts, macrophages, and lymphocytes), has been shown to mediate cellular migration, adhesion, polarity, and cytokinesis via phosphorylating downstream cytoskeleton mediator proteins (84). ROCK suppresses eNOS (NO generation), promotes EndMT, switches VSMCs to a migratory, proliferative phenotype, and regulates M1/2 alternation in macrophages (84). Moreover, ROCK promotes inflammation and OS, since the upregulation of IL-6, TNF-α, IFN-γ, and SOD is accompanied with ROCK overexpression in sepsis models (85). Nonetheless, several drugs have been reported to be ROCK-resistant. Statins, for instance, can suppress RhoA/ROCK via competing TGF-β1/Smad activation, as well as p38-MAPK (86). A study has shown that PAI-1 (plasminogen activator inhibitor-1) mutant mice have higher levels of Col-1 expression and coronary perivascular fibrosis (87). Intriguingly, apelin, an endogenous peptide expressed by ECs, has the capacity to block Ang II induced collagen and MMP-2 expression as well as PAI-1-induced Rho kinase activation, indicating a role in anti-fibrosis (88).

Gender and age perform substantially in the immune response, influencing susceptibility to infections, vaccine response, and function in immune system (137). Aging is linked to the occurrence of long-lasting inflammation as well as a general decrease in immunological function (138).

First of all, there are certain variance in immune cells: NK cells are observed to be higher in male, while plasma cell levels are higher in female (139). The mechanisms within could be related to the prohibit effect of estrogen and progesterone on NK cells (140). Meanwhile, female exerts more activated T cells and B cells than male (141). These differences are claimed to be more significantly impacted through aging. Meanwhile, some research suggests aging to be linked with the increase in monocytes, decrease in T cells, and expression of PBMCs inflammatory markers (142).

Secondly, gene and molecular expression impacts the bias of gender and age difference in immune system. Aging influences pro-inflammatory genes expression, which is severer in male (138). According to recent studies, male experience more IFN-γ receptor activation and MAPK signal induced inflammations (139). They also discovered ZFP36 and DUSP2 are two genes that have the highest level in elder male, which represents weaker adaptive immunity, due to T cell inhibition (139). In addition, we have mentioned a more active T and B cell in female, which could be related to upregulated BAFF/APRIL system in women. While BAFF manages the viability of B cells, APRIL controls cellular proliferation which are essential in humoral immunity (139). Unfortunately, BAFF and APRIL levels lead to their increased risk in autoimmune diseases (143).

Lastly, male and female, young and elder, have distinct sensitivity in inflammation or infection. Aging is linked to the development of chronic inflammation as well as a general decrease in immunological function. Some research suggests that the innate immune system of elderly females is more prone to inflammation than that of elderly males (137). However, women have an enhanced immune response to viral infections than men, resulting in quicker pathogen clearance and increased vaccination effectiveness in females (144). For instance, men have a higher susceptibility to several infectious illnesses, as well as a higher fatality rate (139). After the age of 65, men and women's immune system cells differed the most (141). Older female functions slower in acute reaction or sickness yet shows resistance to certain long-term infections (141). Older men, on the other hand, exhibited higher levels of activity in the innate immune system, the body's generic but faster-reacting defense force (145).

Age as well as gender may additionally impact collagen deposition in different tissues. Increased collagen and fibrin deposition occurred in elderly normal controls when compared to non-elderly controls, and this was even more pronounced in elderly patients with nasal polyps (146), which was further confirmed by studying model organisms such as mice and C. elegans (147). The deposition of collagen fibers after surgery is significantly reduced in older men but not in women (148). These results imply that age and sex can have a major effect on collagen deposition throughout different tissues and that analyzing these differences is vital for developing potent therapies for age-related diseases.

It is eventually our consideration of fibrotic diseases for any heterogeneousness that gender and age may cause. Age-related alterations to the cardiac ECM differ by gender, with elderly male hearts being more fibrotic than female hearts (149). Gender-dependent cardiac fibrosis occurs with age in rats, but the underlying processes remain unknown (150). In those with atrial fibrillation, increasing age and female sex are correlated with a higher incidence of atrial fibrosis (151). We should additionally pay close attention to the fact that the occurrence of greater fibrosis in adult skeletal muscle cells increases dramatically with age, which is associated with TGF-β signaling (152). Similarly, it has been presented that female hormones can ameliorate the advancement of cystic fibrosis in the lung, and there is substantial evidence that estrogen plays an essential role in the development of cystic fibrosis (153). One issue that cannot be overlooked is that aging and diabetes are substantial risk factors for the development of penile fibrosis (154). The above findings emphasize the intricate relationships between age, gender, and the genesis of fibrosis in diverse tissues and disorders. These distinctions can aid the development of tailored and effective fibrotic treatments.

Hypertension is the leading cause of morbidity and mortality worldwide, and is associated with an increased risk of cardiovascular disease. Normally, the heart ejects blood, and the arcus aortae dilate and buffer the systolic and diastolic pressures as they pass to provide appropriate tissue perfusion (155). This mechanism is commonly accepted to be strongly dependent on artery elasticity and compliance, whereas vascular fibrosis-induced arterial stiffness lacks this feature (1). It is especially unsettling since hypertension being a direct outcome of arterial remodeling and fibrosing, may be easily obtained through collagen deposition (36). The expression and proliferation of VSMCs can also lead to the thickening of the vessel wall, which enhances transverse aortic contraction and blood pressure (48).

There are several medications that alleviate hypertension through various methods, some of which grabbed our attention due to their anti-fibrosis properties. Firstly, alamandine can reduce blood pressure by preventing Ang II-induced arterial remodeling (36). Secondly, statins, as the inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, have been found to be pleiotropically beneficial against fibrosis and hypertension through inhibiting TGF-β/Smad (86), in addition to its well-known cholesterol-lowering activity. Their anti-fibrotic role leaves us a novel target for resistant hypertension.

Pulmonary arterial hypertension (PAH) is one particular hypertension in the lungs, defined as an ≥20 mmHg resting pulmonary arterial pressure in mean (156). Pulmonary illness and dyspnea are inseparable from PAH, partly due to pulmonary artery smooth muscle cell (PASMC) proliferation, inflammation, and severe fibrosis (73). Scholars show special interest in their latest discovery in a cluster of PAH-related mediators, including gal-3, StAR, caspase-1, and programmed death ligand-1 (PDL-1). PDL-1, along with caspase-1, is implicated in pyroptosis, which is associated with the production of fibrosis and PAH (58). Furthermore, the research found that NEDD9, a TGF-β independent mediator, can trigger fibrosis in PAH via oxidative modification at Cys 18 (157).

Atherosclerosis, one of the leading causes of CVD, is characterized by impaired degradation of ECM (collagen and fibronectin), VSMCs proliferation, as well as calcification (49), therefore lies connection with fibrosis. EndMT has been widely discovered in atherosclerosis due to its promotive effect in plaque formation. EndMT generates mesenchymal cells that produce pro-inflammatory cytokines, ECM proteins, and MMPs (158). As research deepens, certain chemicals strongly linked to atherosclerosis have been discovered. For example, gal-3 overexpresses in plaque, salusin-β TGF-β-dependently promotes Col-1/3 expression, and ncRNAs (miR-125b, miR-21, Let-7c, Let-7g) generally expresses in the thickened vascular wall (35, 49, 103). Intriguingly, a study on a natural substance paeonol revealed that it has a role in atherosclerosis suppression by enhancing gut microorganisms' short-chain fatty acids (SCFAs) synthesis to modulate Treg/Th17 balance in the spleen, hence preventing vascular fibrosis (159). Statins also alleviates atherosclerosis by down-regulating the expression of Ang II-induced profibrotic factors and ECM proteins (86). Furthermore, H₂S and specific microRNAs (miR-483, miR-126, miR-155) have a certain role in alleviating vascular fibrosis and atherosclerosis. The experimental evidence shown above illustrates that the occurrence, progression, and therapy of atherosclerosis is a tremendously complicated process involving different routes and cytokines or active substances.

As mentioned above, vascular inflammation is highly associated with fibrosis. Takayasu's arteritis (TAK) is a chronic inflammatory disease characterized by granulomatous vasculitis and fibrosis in the aorta and its branches (47). Perfusion is hampered by persistent inflammation and fibrosis, which leads to organ failure and death (160). Multiple fibrotic biomarkers, including TGF-β, Col-1/3, fibronectin, and α-SMA, have been found elevated in TAK arteries than in normal arteries (161). Unfortunately, conventional therapies with glucocorticoids and immunosuppressants can only stabilize inflammation, but behaved badly against existing fibrosis-induced structural damage. Research has revealed that vascular fibrosis in TAK is promoted by IL-6 related autophagy via JAK1/STAT3 pathway, implying impeding autophagy, as well as IL-6 inhibitor, may be beneficial in the treatment of TAK (161, 162). Leflunomide (LEF), an anti-inflammatory medication, has been demonstrated to diminish TAK by blocking and lowering macrophage subtype M2 rather than M1 (160). Another study found that Curcumin and Etomoxir exhibit properties of anti-fibrosing, targeting FABP3 to modulate FAO in AAFs as a possible treatment for TAK and other vascular fibrosing conditions.

Furthermore, vascular fibrosis is found in Kawasaki disease (KD), a well-documented but poorly understood systemic pediatric vasculitis characterized by multisystem inflammatory syndrome (MIS), severe toxic shock syndrome, and coronary artery aneurysm (163, 164). In KD model mice, coronary artery inflammation pyroptosis-related factors (NLRP3, caspase-1, IL-1β, IL-18) exhibited a significant upregulation in ECs (59), showing EC pyroptosis participates in KD. Intriguingly, SARS-CoV-2 was identified to coincide with KD in MIS-C degree (163, 165), hence understanding the mechanisms of SARS-CoV-2 may be the potential key to unmasking KD. In addition, studies have demonstrated that gal-3 and TGF-β/Smad levels are much higher in KD patients, making them appealing therapeutic targets for KD and fibrotic dysfunction (166, 167).

Evidence has confirmed that there is a linkage between vascular fibrosis and endocrine disturbance. Diabetes, particularly type 2 diabetic mellitus (DM2), that shares consistency with vascular fibrosis according to multiple studies. On one hand, fibrosis directs DM: Experiencing ECM deposition, cytoarchitectural abnormalities, microvascular dysfunction, and impaired endocrine cell-blood exchanges in pancreatic islets lead to aberrant insulin secretion (168). Cell type modifications, such as the conversion of pericytes to fibroblasts and EndMT, are critical in triggering DM2 vascular fibrosis (49, 168). DM is also linked to autonomous cortisol secretion (ACS) in individuals with primary aldosteronism (PA) (169). PA patients with ACS had a considerably greater diabetes incidence and a larger fibrosis area in blood vessels, indicating that an ACS show-up implies poor vascular remodeling (169). On the other hand, DM2 lures fibrosis in aortic or other cardiovascular tissue: developing DM boosts myofibroblasts activation, inhibits macrophage M2 polarization, induces a profibrotic phenotype in immune cells, increases ROS, and initiates EndMT (81, 170, 171). Interestingly, various fibrotic-associated molecular alterations were observed in salt-loaded DM2 mice, including increased TGF-β, Col-1, and fibronectin, as well as decreased Fli-1, which could be reversed by MBG (81, 83). In salt-loaded DM2 patients, experiencing a Na⁺–K⁺–ATPase malfunction might be the cause of vascular injury and remodeling. MBG, as its inhibitor, contributes to this pathological condition by suppressing it, therefore benefiting DM2 (81). In addition, rosiglitazone as a classic drug on insulin resistance DM2 patient, have attached repressive impact on Ang II-induced AT-1R, IL-6, and TNF-α elevation (172), as well as VSMCs and myofibroblasts proliferation (173, 174). These changes vastly benefit vascular fibrosis. Another study indicates rosiglitazone, as a PPAR-γ activator, benefits in DM2 by lowering CTGF expression and ECM deposition in Ang II-induced fibrosis (175). However, along with other thiazolidinediones (TZDs), rosiglitazone may be associated with severe side effects including heart failure and pulmonary edema (176), therefore its clinical application is restricted and must be paid particular attention. Nevertheless, many research suggest rosiglitazone attenuative to serum level of fibronectin, TGF-β, PAI-1, and inflammatory marker (MMP-2, MMP-9, COX-2) in DM2 patients (177, 178). Based on the evidence presented above, we may deduce that there is a pertinent association between diabetes mellitus and vascular fibrosis, and that this link impacts the prognosis and progression of the disease, while also providing a novel target for vascular fibrosis treatment.

Since myocardial fibrosis is frequently coupled with vascular fibrosis, both molecular mechanisms and pathological processes are intertwined. Chronic hypertension and arteriosclerosis increase cardiac afterload, leading to myocardial hypertrophy, coronary heart disease, heart failure, and other complications. In the aging kidney, T and B cells, together with resident fibroblasts, form tertiary lymphoid tissues that cause uncontrollable inflammation and delay tissue repair (179). Furthermore, severe fibrosis of small blood vessels can cause parenchymal tissues and cells to grow, leading to fatal diseases including liver cirrhosis and brain fibrosis (58). However, fibrosis is not a one-way road. To slow the process of organ fibrosis, ECM protein degradation, chronic inflammation regression, tissue injury resolution, and myofibroblasts deactivation are all required (180). Currently, a number of anti-fibrosis medications are being tested in clinical trials. Nintedanib is an antifibrotic drug licensed for patients with idiopathic pulmonary fibrosis (IPF) by blocking several tyrosine kinases implicated in fibrosis development (181). Another anti-fibrotic medicine, Pirfenidone, has been licensed for use in individuals with IPF to decrease disease development by reducing the production of pro-fibrotic cytokines and growth factors (182). It should be emphasized that lowering oxidative stress can also help slow or stop fibrosis progression (183). As previously stated, some immune modulation is involved in the genesis of fibrosis (184).