Since 2000, Nebigil et al. have suggested that the 5-HT_2BR is implicated in regulating cardiac structure and function during embryogenesis and adulthood [9]. The ablation of the 5-HT_2BR in mice led to embryonic and neonatal death. Surviving 5-HT_2BR knockout mice exhibited cardiomyopathy with decreased cardiomyocyte number and size. On the contrary, specifically overexpressing the 5-HT_2BR in the heart led to compensated hypertrophic cardiomyopathy, characterized by ventricular wall thickening [11].

Numerous animal model studies further confirmed the role played by the 5-HT_2BR in cardiomyopathy. The 5-HT_2BR has been found to be associated with isoproterenol- and noradrenaline-induced cardiac hypertrophy [12,13,14]. Chronic isoproterenol perfusion in mice imitating sympathetic stimulation induced cardiac hypertrophy, which could be prevented by treatment with 5-HT_2BR antagonists, through regulating the hypertrophic cytokines produced by cardiac fibroblasts [12] and the production of superoxide anion [13]. In rats, a 5-HT_2BR antagonist attenuated cardiac hypertrophy and myocardial apoptosis induced by chronic noradrenaline treatment [14]. In dogs with dilated cardiomyopathy, the 5-HT_2BR was overexpressed in cardiomyocytes [15].

The normal mammalian heart has four valves to ensure unidirectional blood flow during the cardiac cycle: the mitral valve (from the left atrium to the left ventricle), the tricuspid valve (from the right atrium to the right ventricle), the aortic valve (from the left ventricle to the aorta), and the pulmonary valve (from the right ventricle to the pulmonary artery). Any damaged or diseased heart valve can result in VHD. Abnormal valves cannot be fully open (stenosis) or fully close (regurgitation) so that the blood cannot be effectively pumped throughout the body, resulting in heart failure, sudden cardiac arrest and even death in more severe cases. Fully formed heart valves consist of valvular endothelial cells and valvular interstitial cells (VICs). The two types of cells regulate the generation of the extracellular matrix (ECM) and thus play critical roles in valve function [2]. Excessive ECM alters valve structure and leads to VHD.

Several drugs are known to associate with VHD side effects, including therapeutic agents for the treatment of obesity (fenfluramine and its stereoisomer dexfenfluramine, and benfluorex), Parkinson’s disease (pergolide and cabergoline), and migraine (methysergide and ergotamine), as well as the recreational drug 3,4-methylenedioxymethamphetamine (MDMA, commonly known as ecstasy) [16,17]. Drug-induced VHD has led to the withdrawal of fenfluramine and dexfenfluramine from the U.S. market in 1997, followed by the withdrawal of pergolide in 2007. Either these drugs or their metabolites have been demonstrated to be partial or full 5-HT_2BR agonists with high affinity, and the pathogenesis of drug-induced VHD was correlated to the “off-target” activation of the 5-HT_2BR [2,18]. Consequently, drug candidates with possible 5-HT_2BR agonism effects are now required to be evaluated before approval [19]. Additionally, the signaling mechanism of drug-induced VHD has been studied [2,20]. Apart from the canonical G_q/11 signal transduction pathway involved in the activation of PLCβ and PKC, the activation of the 5-HT_2BR may also activate mitogenic pathways through the phosphorylation of the Src kinase and extracellular regulated kinases (ERK) and further enhance the activity of the transforming growth factor β (TGF-β). All pathways lead to VIC proliferation and ECM accumulation, and subsequently to the occurrence of VHD.

The 5-HT_2BR was shown to be involved in vascular heart diseases, including mitral valve prolapse (MVP) [21] and calcific aortic valve disease (CAVD) [22,23]. Overexpression of the 5-HT_2BR in the mitral valve leaflets was found in humans with MVP. Blockade of the 5-HT_2BR mitigated mitral valve thickening and the activation of mitral valve interstitial cells, which are involved in the pathophysiology of MVP [21]. A study in isolated aortic valve interstitial cells (AVICs) in vitro showed that 5-HT_2BR antagonism could prevent AVIC activation, a process associated with CAVD [22]. Recently, the same research group reported that in a high cholesterol diet mouse model, aortic valve hemodynamic development of CAVD could be attenuated by the ablation of the 5-HT_2B gene, but not 5-HT_2BR antagonism [23].

PAH is a progressive disorder characterized by abnormally high blood pressure in pulmonary arterial and pulmonary vasculature remodeling. The involvement of the 5-HT_2BR in PAH has long been suggested. A significantly increased expression of the 5-HT_2BR in pulmonary arteries was found in pulmonary hypertension (PH) patients and mice [24]. Moreover, the upregulation of the 5-HT_2BR has been found in pulmonary artery smooth muscle cells derived from PAH patients [25]. In vivo studies on animal models suggested that chronic hypoxia or chemicals, such as deoxycorticosterone acetate (DOCA) salt and monocrotaline (MCT), could induce PH, which can be prevented or alleviated through blocking the 5-HT_2BR or by genetic ablation [24,25,26,27]. In a BMPR2 mutant imitating heritable PAH mouse model, 5-HT_2BR antagonism prevents PAH through reducing Src phosphorylation and downstream activity [28].

Emerging evidence has shown that bone marrow (BM)-derived cells contribute to 5-HT_2BR-mediated PAH. Launay et al. found that lung cells overexpressing 5-HT_2BRs for vascular remodeling during PAH originate from BM precursors in mice [29]. They found that the specific expression of 5-HT_2BR in the BM is necessary and sufficient for PAH development, whereas the ablation of 5-HT_2BR on BM cells leads to resistance to PH. More recently, Bloodworth et al. demonstrated that BM-derived proangiogenic cells play a role in PH by mediating pulmonary arteriole stiffening and remodeling via the 5-HT_2BR [30]. Both the ablation of BM-derived proangiogenic cells and 5-HT_2BR antagonism prevented PH in mice with reductions in the number and stiffness of muscularized pulmonary arterioles.

Liver fibrosis is generally believed to be caused by the excessive production of ECM, which is promoted by activated hepatic stellate cells (HSCs) transdifferentiating into myofibroblasts [45,46]. Ebrahimkhani et al. found that the 5-HT_2BR was highly expressed in the diseased liver by activated HSCs and that 5-HT_2BR antagonism exerted an antifibrogenic effect and improved liver function in a mouse model of progressive liver disease with fibrogenesis [31]. In a carbon tetrachloride (CCl₄)-induced liver fibrosis mouse model, Li et al. found that chronic restraint stress alleviated liver fibrosis by inhibiting the activation of HSCs via the 5-HT_2BR [32]. More recently, Xiang et al. revealed that two microRNAs (miR-221 and miR-222) were regulated by 5-HT during HSC activation and that the 5-HT_2BR was essential for this regulation, as demonstrated by the discovery that 5-HT did not increase the expression of miR-221/miR-222 in 5-HT_2B knockdown HSCs [33].

Pulmonary fibrosis is one of the most studied 5-HT associated fibrosis. Fibroblasts (effector cells) differentiate into myofibroblasts and subsequently synthesize ECM, which are considered key events in pulmonary fibrogenesis. Fabre et al. found that the 5-HT_2BR was highly expressed by fibroblasts in the fibroblastic foci in human idiopathic pulmonary fibrosis (IPF) samples [34]. In the lungs of IPF patients, Königshoff et al. found that the 5-HT_2BR mainly localized to the epithelium and showed a significant increase in expression compared to transplant donors [35].

In vivo studies in the bleomycin (BLM)-induced pulmonary fibrosis mouse model suggested the involvement of 5-HT_2AR and 5-HT_2BR in pulmonary fibrosis. The expression of 5-HT_2AR and 5-HT_2BR were increased in the lung after the intratracheal treatment with BLM [34,35]. Blockade of the 5-HT_2AR and 5-HT_2BR could ameliorate BLM-induced lung fibrosis and improve lung function by reducing lung collagen content [34,35]. In vitro studies in human lung, fibroblasts showed that the antifibrotic effect of 5-HT_2AR and 5-HT_2BR antagonism was mediated by the TGF-β1 and WNT3α signaling pathways [35]. Moreover, Löfdahl et al. utilized two 5-HT_2BR antagonists EXT5 and EXT9 (also with low to moderate affinity to the 5-HT_2A/5-HT_2C receptors), to investigate the role of the 5-HT_2BR in pulmonary fibrosis, suggesting their potential to prevent myofibroblast differentiation and subsequent fibrotic responses in a BLM-treated mouse model and human lung fibroblasts (see Section 4.2.1 for more details) [36]. Further studies suggested that the antiproliferative effects of EXT5 and EXT9 were related to the pAkt/p21 signaling pathway [38]. It is worth mentioning that in vivo studies in the BLM-induced pulmonary fibrosis rat model showed that 5-HT_2CR and 5-HT₇R were also implicated in pulmonary fibrosis [37,47,48].

SSc is a chronic autoimmune disease characterized by progressive vascular disease and fibrosis of the skin and internal organs. Emerging evidence has suggested that 5-HT_2BR plays an important role in SSc. In 2011, Dees et al. found that the expression of the 5-HT_2BR was significantly increased in the skin of SSc patients compared with the normal skin of healthy individuals [39]. In vitro studies on SSc dermal fibroblasts suggested that the profibrotic effects of 5-HT are mediated by the 5-HT_2BR, excluding the 5-HT_1BR and 5-HT_2AR, which are also expressed in dermal fibroblasts [39]. In vivo studies on BLM-induced dermal fibrosis and tight-skin-1 (tsk-1) mouse models showed that 5-HT_2BR antagonists ameliorated fibrosis. In addition, mice lacking the 5-HT_2B gene could be protected from BLM-induced fibrosis [39]. In 2018, Chaturvedi et al. studied human adult dermal fibroblasts (HADF) isolated from SSc patients and showed that stimulation of 5-HT/TGF-β1 in HADF significantly increased the expression of profibrotic genes. Profibrotic genes were downregulated by the 5-HT_2BR antagonist SB-204741, whose antifibrotic effect might be involved in the suppression of TGF-β1-mediated non-canonical (non-Smad dependent) signaling pathways [40]. Moreover, Wenglén et al. discovered selective antagonists of the 5-HT_2BR (AM1125 and AM1476) and suggested their antifibrotic effects for the potential treatment of SSc (see Section 4.2.1 for details) [42,43].

Through the analysis of liver tissues from patients with HCC, Sarrouilhe et al. found that the 5-HT_1BR and the 5-HT_2BR were overexpressed in tumor tissues and that their antagonists inhibited proliferation of HCC cell lines, such as Huh7 and HepG2 [50]. Liang et al. suggested that 5-HT promoted the proliferation of serum-deprived Huh7 cells by upregulating the transcription factor FOXO3a, although this pro-proliferative effect was not observed in serum-deprived HepG2 or Hep3B cells [51]. They further found that the pro-proliferative effect of 5-HT could be blocked by the 5-HT_2BR antagonist SB-204741 in Huh7 cells and that 5-HT_2BR mRNA was significantly higher expressed in Huh7 cells compared to HepG2 and Hep3B cells, which may contribute to the distinct 5-HT effects in different serum-deprived HCC cells [51]. Using zebrafish HCC models, Yang et al. suggested that the 5-HT_2BR was involved in HCC carcinogenesis [52,53]. In zebrafish, the expression of the 5-HT_2BR was found to be high in HSCs, much lower in hepatocytes, and practically absent in neutrophils and macrophages [53]. The activation of the 5-HT_2BR could increase both the proliferation and the activation of HSCs, as well as the expression of TGF-β1, resulting in liver enlargement and accelerating HCC carcinogenesis. In contrast, blocking the 5-HT_2BR led to opposite effects [52,53].

NET is a rare type of tumor that most commonly arises in the GI tract and can lead to carcinoid syndrome [54,55]. Svejda et al. studied KRJ-I cells, a small intestinal-NET (SI-NET) cell line, and found that treatment with 5-HT_2BR antagonist PRX-08066 inhibited the 5-HT secretion and KRJ-I cell proliferation, simultaneously decreasing the phosphorylation of ERK1/2 and the transcript levels and secretion of profibrotic growth factors, including TGF-β1, connective tissue growth factor (CTGF) and fibroblast growth factor (FGF2). The antiproliferative and antifibrotic effects of the 5-HT_2BR suggested that this is a promising target for intervening SI-NETs [56].

In 2017, Jiang et al. reported that the 5-HT_2BR could be used as a potential therapeutic target for intervening pancreatic ductal adenocarcinomas (PDACs) [57]. 5-HT was found to be increased in human PDAC tissues. Moreover, the incubation of 5-HT with PDAC cell lines resulted in an increase in PDAC cell proliferation and a decrease of PDAC cell apoptosis. Both in vitro and in vivo studies demonstrated that the pro-survival effect of 5-HT is mediated by the 5-HT_2BR, but not other 5-HTRs. The 5-HT_2BR agonist α-Me-HTP promoted the survival of PDAC cells, whereas the 5-HT_2BR antagonist SB-204741 or genetic silencing of the 5-HT_2BR blocked the pro-survival effect of 5-HT in PDAC cells and significantly reduced the tumor burden of PDAC in mice [57]. Moreover, the tumor-suppressive effects of 5-HT_2BR antagonism were further confirmed in transgenic mice with pancreatic tumors. Notably, the mechanism behind 5-HT mediated PDAC cell survival involved the activation of PI3K/Akt/mTOR signaling and the enhancement of aerobic glycolysis (Warburg effect) [57].

The 5-HT_2BR has been implicated in migraine and neuropathic pain, which are two common forms of pain disorders in humans [75,76,77]. Migraine is a common primary headache disorder characterized by moderate to severe recurrent headaches. In 1989, Fozard et al. proposed that the initiation of migraine is caused by the activation of the 5-HT_2CR [78]. However, this hypothesis was challenged after the cloning of rat 5-HT_2BR in 1992 [4]. Subsequent studies demonstrated that the 5-HT_2BR activation stimulated nitric oxide (NO) synthesis, which may be involved in migraine pathogenesis [75]. In guinea pigs, selective 5-HT_2BR antagonists have been found to inhibit the 5-HT_2BR/5-HT_2CR agonist meta-chlorophenylpiperazine (mCPP) or the 5-HT_2BR agonist BW723C686-induced dural plasma protein extravasation (PPE), an indicator for migraine attacks in animal models [79]. In addition, 5-HT_2BR antagonism also prevented mCPP-induced dural PPE under hypoxia in mice [80].

Increasing evidence has revealed that the 5-HT_2BR also plays a role in neuropathic pain [77]. In mouse dorsal root ganglion (DRG) neurons, the mechanical hyperalgesia induced by 5-HT or the 5-HT₂R agonist α-m5-HT was inhibited by the 5-HT_2BR/5-HT_2CR antagonist SB-206553 [81]. Given that the 5-HT_2BR was mainly expressed in DRGs, whereas the 5-HT_2CR was detected only in trace amounts, 5-HT-induced mechanical hyperalgesia is most likely mediated by the 5-HT_2BR [81]. Another signal transduction study suggested that the 5-HT_2BR mediates the 5-HT-induced mechanical hyperalgesia through the PLCβ-PKCε pathway to regulate the function of transient receptor potential vanilloid 1 [82]. Cervantes-Durán et al. assessed the role played by peripheral and spinal 5-HT₂Rs in formalin-induced secondary allodynia and hyperalgesia in rats. Local peripheral ipsilateral or intrathecal injection of selective 5-HT_2BR antagonist significantly prevented formalin-induced nociceptive behavior monitored by flinching frequency [83]. Ipsilateral treatment with subtype-selective antagonists of 5-HT_2AR, 5-HT_2BR or 5-HT_2CR, prevented formalin-induced long-term secondary mechanical allodynia and hyperalgesia [84]. Additionally, intrathecal treatment with the same antagonists inhibited formalin-induced long-term secondary mechanical allodynia and hyperalgesia in both ipsilateral and contralateral hind paws [85]. In the spinal nerve ligation-induced neuropathic pain rat model, intrathecal injection of 5-HT_2BR antagonists not only impaired spinal nerve ligation-induced allodynia but also inhibited the spinal nerve injury-induced increased expression of the 5-HT_2BR in both DRGs and spinal cord [86]. More recently, studies in female rats revealed that blocking the spinal 5-HT_2BR diminished preoperative anxiety-induced postoperative hyperalgesia [87]. However, opposite findings were reported in other pain models. For example, in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve, Urtikova et al. found that intrathecal injection of the 5-HT_2BR agonist BW723C86 evidently relieved CCI-induced allodynia [88]. Clearly, further mechanistic studies are needed to explain the opposite experimental observations.

The 5-HT_2BR is also expressed in neuroglia, including microglia and astroglia, playing a role in regulating neuroglia function. Microglia, as the resident macrophages in the brain and the spinal cord, are responsible for the immune defense of the central nervous system (CNS) [89]. It was reported that the 5-HT_2BR was expressed on postnatal microglia and participated in postnatal brain maturation [90]. More recently, the same group showed that the ablation of the 5-HT_2BR gene in neonatal microglia was sufficient to cause enhanced weight loss and prolonged neuroinflammation in mice caused by exposure to lipopolysaccharides in adulthood. This suggested that the 5-HT_2BR is required in neonatal microglia to prevent sickness behavior in adulthood [91].

Astrocytes are primary homeostatic cells of the CNS and account for about one-quarter of brain cortical volume. The expression of 5-HTRs, including the 5-HT_2BR, has been found in both cultured and freshly isolated astrocytes [92]. Studies have suggested that conventional serotonin-specific reuptake inhibitors (SSRIs) such as fluoxetine act as agonists of astroglial 5-HT_2BR [92]. The 5-HT_2BR agonist BW723C86 could mimic the behavioral and neurogenic SSRI effects, which could be eliminated by the genetic or pharmacological inactivation of the 5-HT_2BR [93]. In cultured mouse astrocytes, fluoxetine was found to induce EGFR transactivation and ERK1/2 phosphorylation, mediated by the stimulation of the 5-HT_2BR [94], which is consistent with the observation that the drug-induced VHD involves the activation of the 5-HT_2BR and consequent ERK phosphorylation [2,20]. Similarly, 5-HT was also found to cause ERK1/2 phosphorylation, which is mediated by the stimulation of the 5-HT_2BR with high affinity and the 5-HT_2CR with low-affinity [95]. Increasing evidence has suggested that astroglial 5-HT_2BR is involved in depression [96]. In both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced and 6-hydroxydopamine-induced Parkinson’s disease mouse models [97,98], the decrease of astroglial 5-HT_2BR expression paralleled the development of depressive behavior. Treatment with fluoxetine corrected both the decrease of astroglial 5-HT_2BR expression and depressive behavior. All of these indicate that the downregulation of the astroglial 5-HT_2BR may promote the development of depressive behavior in Parkinson’s disease. In addition, astroglial 5-HT_2BR was also found to play a role in depressive behavior associated with sleep deprivation [99,100]. Specifically, the expression of the 5-HT_2BR in a sleep deprivation mouse model was downregulated selectively in astrocytes, which was controlled by the activation of the P2X7 receptor [99]. Interestingly, leptin was found to increase the expression of astrocytic 5-HT_2BRs and thus enhance the action of fluoxetine on depressive-like behaviors induced by sleep deprivation [100].