Heart Failure Reviews (2023) 28:555–569 https://doi.org/10.1007/s10741-022-10279-x Treatment of cardiac fibrosis: from neuro‑hormonal inhibitors to CAR‑T cell therapy Paolo Morfino1 · Alberto Aimo1,2 · Vincenzo Castiglione1 · Carolina Gálvez‑Montón3,4 · Michele Emdin1,2 · Antoni Bayes‑Genis3,4,5 Accepted: 3 October 2022 / Published online: 11 October 2022 © The Author(s) 2022 Abstract Cardiac fibrosis is characterized by the deposition of extracellular matrix proteins in the spaces between cardiomyocytes following both acute and chronic tissue damage events, resulting in the remodeling and stiffening of heart tissue. Fibrosis plays an important role in the pathogenesis of many cardiovascular disorders, including heart failure and myocardial infarc- tion. Several studies have identified fibroblasts, which are induced to differentiate into myofibroblasts in response to various types of damage, as the most important cell types involved in the fibrotic process. Some drugs, such as inhibitors of the renin–angiotensin–aldosterone system, have been shown to be effective in reducing cardiac fibrosis. There are currently no drugs with primarily anti-fibrotic action approved for clinical use, as well as the evidence of a clinical efficacy of these drugs is extremely limited, despite the numerous encouraging results from experimental studies. A new approach is represented by the use of CAR-T cells engineered in vivo using lipid nanoparticles containing mRNA coding for a receptor directed against the FAP protein, expressed by cardiac myofibroblasts. This strategy has proved to be safe and effective in reducing myocardial fibrosis and improving cardiac function in mouse models of cardiac fibrosis. Clinical studies are required to test this novel approach in humans. Keywords Fibrosis · Myocardium · Anti-fibrotic therapies · Heart failure · CAR-T cells Fibrosis results from excessive deposition of extracellular Tissue repair mechanisms that lead to the development matrix (ECM) proteins, especially type I and III collagen, of fibrosis are activated by acute or chronic tissue damage. between tissue parenchymal cells. There are conditions char- The inflammatory response can be intense depending on acterized by an activation of the pro-fibrotic pathways sec- the type of damage; it favors the release of mediators and ondary to other disease mechanisms, such as neuroendocrine the migration of neutrophils, eosinophils, and macrophages activation in heart failure (HF), and conditions in which the to the injured site. Tissue fibroblasts and other mesenchy- first cause of disease is the deposition of fibrous tissue, such mal cells are activated, differentiating into myofibroblasts, as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis which are cells with contractile action and secreting activity [1, 2]. In both cases, fibrosis can lead to organ dysfunction. of ECM elements, when stimulated by pro-fibrotic cytokines such as fibroblast growth factor (FGF), platelet-derived * Alberto Aimo growth factor (PDGF), and transformant-β growth factor a.aimo@santannapisa.it; aimoalb@ftgm.it (TGF-β) [3]. Myofibroblasts may also acquire resistance to 1 cell death stimuli [4]. At the same time, the expression of Interdisciplinary Center for Health Sciences, Scuola Superiore Sant’Anna, Pisa, Italy matrix metalloproteinases (MMPs) is reduced. MMPs are 2 mainly responsible for the replacement and degradation of Cardiology Division, Fondazione Toscana Gabriele Monasterio, Pisa, Italy ECM proteins [5]. 3 Myocardial fibrosis is traditionally distinguished into Institut del Cor, Hospital Universitari Germans Trias I Pujol, Badalona, Spain “reparative” or “reactive” (Fig. 1). Reparative fibrosis is 4 the replacement of necrotic cardiomyocytes following events CIBERCV, Carlos III Institute of Health, Madrid, Spain of acute and extensive tissue damage, such as myocardial 5 Department of Medicine, Universitat Autònoma de infarction (MI) or myocarditis. Other forms of myocardial Barcelona, Barcelona, Spain 13 Vol.:(0123456789) 556 Heart Failure Reviews (2023) 28:555–569 Fig. 1  Types of cardiac fibrosis. The extracellular matrix in the “Reactive” fibrosis (right) accompanies heart failure and pressure healthy heart (left) is a three-dimensional network of collagen fibrils overload, and manifests as diffuse collagen deposition in interstitial that incorporates cardiomyocytes, capillaries, and fibroblasts. The and perivascular areas. Modified with permission from Schimmel “reparative” fibrosis (center) is visible as a collagen-based scar that et al. [115] replaces necrotic cardiomyocytes after acute and extensive damage. damage result in reactive fibrosis, with peculiar ECM struc- (ECV) [14, 16]. Regarding in vivo investigation techniques, ture and composition depending on the pathogenic cause of the assay of circulating biomarkers of fibrosis is largely damage (Fig. 2). For example, pressure overload is charac- adopted. Among biomarkers of fibrosis, we can find col- terized by perivascular fibrosis and interstitial fibrosis due lagen fragments and precursors, representing synthesis to the activation of pro-fibrotic pathways (angiotensin II and biomarkers; MMPs and MMP tissue inhibitors (TIMPs), endothelin 1) [6, 7]. The two forms of fibrosis often develop representing degradation biomarkers; and galectin-3 and together within the same disease: reparative fibrosis follow- soluble tumor-2 suppressor [17]. However, recent studies ing MI represents the immediate tissue response to damage; show that the increase in plasma collagen biomarkers does then, hormonal and paracrine activation trigger the reactive not really reflect the extent of myocardial fibrosis, suggest- fibrosis in remote regions at MI, leading to post-infarction ing that new studies are needed for the validation of fibrosis ventricular remodeling [8]. Myocardial fibrosis also slows biomarkers [18]. the conduction of the cardiac action potential and predis- The amount of cardiac fibrosis has an important clini- poses to conduction disorders such as atrioventricular block cal and prognostic value. For example, there is a relation- [9]. Fibrotic regions also promote the generation of re-entry ship between fibrotic extension in the left atrium at CMR circuits and can trigger focal arrhythmias. In addition, myo- and the efficacy of transcatheter ablation in patients with cardial fibrosis determines a chaotic and non-linear prop- AF, with a greater recurrence of AF in subjects showing agation of the action potential. Fibrotic infiltration in the a larger pre-ablation fibrotic area [19]. In subjects with atrium promotes the onset of atrial fibrillation (AF), as well hypertrophic cardiomyopathy, the presence of fibrosis at as impairs the compliance and thus the mechanical proper- CMR led to a 3.4-fold higher risk of mortality or hospi- ties of the atrium [10, 11]. In addition, atrial fibrosis may talization due to cardiovascular causes or implantation of contribute to the development of atrial amyloidosis since cardioverter-defibrillator, with a 15% increase in risk as a the accumulation of amyloid substance is accompanied by result of a 5% increase in fibrosis; whereas in subjects with the deposition of fibrous tissue, as observed in the ventricles acute coronary syndrome the presence of cardiac fibrosis at [12, 13]. CMR is strongly associated with sudden cardiac death [20, Histological analysis of endomyocardial biopsy samples 21]. For these reasons, myocardial fibrosis can be considered with trichromatic and Sirius Red staining allows a direct an important therapeutic target. evaluation of cardiac fibrosis [14, 15]. A non-invasive alter- Some drugs, such as renin–angiotensin–aldosterone native for detecting myocardial fibrosis in vivo is cardiac system (RAAS) inhibitors, have been shown to be effec- magnetic resonance (CMR) imaging. Fibrotic regions are tive in reducing ECM deposition in the myocardium. identified as late gadolinium enhancement areas. RMC However, no primarily anti-fibrotic medications have also allows for the quantification of extracellular volume been approved for the treatment of cardiovascular disease 13 Heart Failure Reviews (2023) 28:555–569 557 Fig. 2  Schematic representation of heterogeneity in fibrotic progres- metalloproteinase; MV, microvessel; N, neutrophil; PAI, plasminogen sion. (Illustration: Maartje Kunen, Medical Visuals.) AngII, angio- activator inhibitor; PDGF, platelet-derived growth factor; TGF, trans- tensin II; CTGF, connective tissue growth factor; DAMPS, danger- forming growth factor; TIMP, tissue inhibitor of metalloproteinase; associated molecular patterns; ET-1, endothelin-1; IL, interleukin; TNF, tumor necrosis factor. Reprinted with permission by de Boer L, lymphocyte; Ma, macrophage; MC, mast cell; MCP-1, monocyte et al. [14] chemoattractant protein-1; MF/MyoF, myofibroblast; MMP, matrix [22]. Studies on animal models have revealed a wide vari- Drugs with no primarily anti‑fibrotic role ety of effective molecules in the treatment of fibrosis, but there is still no evidence of clinical benefit (Table 1). RAAS inhibitors Recently, the first results from the phase II clinical trial PIROUETTE (The Efficacy and Safety of Pirfenidone in Angiotensin II (Ang II) binding Ang II type 1 receptors Patients with Heart Failure and Preserved Left Ventricu- (AT1R) promotes collagen synthesis [25]. In chronic lar Ejection Fraction) show that pirfenidone may be effec- heart disease, there is generally a significant activation tive and safe in patients with HF and preserved ejection of RAAS, which is directly associated with the develop- fraction (FE) [23]. Anti-fibrotic T-cell therapy with chi- ment of cardiac fibrosis [26]. Various studies have shown meric antigen receptor (CAR) is widely used in oncology. that both angiotensin-converting enzyme (ACE) inhibi- Recent studies on preclinical models also encourage their tors and angiotensin II receptor blockers (ARBs) sig- potential use in cardiovascular diseases [24]. nificantly reduce myocardial fibrosis regardless of their This literature review summarizes the possible options hypotensive effect. In a sample of hypertensive patients to target myocardial fibrosis, including the new perspec- treated with lisinopril (n = 18), endomyocardial biopsy tive of CAR-T cell therapy. at 6 months revealed a significant reduction in collagen 13 558 Heart Failure Reviews (2023) 28:555–569 Table 1  Main evidence of anti-fibrotic drugs from clinical studies Study Therapy Follow-up Included Main evidence patients (n) RAAS inhibitors Brilla et al. [27] Lisinopril 6 months 35 Lisinopril reduces CVF in hypertensive patients compared with hydrochloro- thiazide diuretic López et al. [28] Losartan 12 months 37 Losartan reduces CVF and PICP in hypertensive patients compared with amlodipine Díez et al. [29] Losartan 12 months 19 Losartan reduces CVF and LV stiffness in hypertensive patients with severe fibrosis Shimada et al. [30] Losartan 12 months 20 Losartan reduces fibrotic progression in patients with nonobstructive hyper- trophic CMP Kosmala et al. [32] Spironolactone 6 months 80 ACEi and spironolactone reduce PICP and PIIINP in patients with metabolic syndrome compared with ACEi alone Kosmala et al. [33] Spironolactone 6 months 113 Spironolactone improves myocardial deformation and reduces PICP and PIIINP in obese patients with mild diastolic dysfunction Mak et al. [34] Eplerenone 12 months 44 Eplerenone reduces PIIINP and slightly improves diastolic function in patients with diastolic HF Deswal et al. [35] Eplerenone 6 months 44 Eplerenone reduces PINP and PICP in patients with HFpEF Zannad et al. (post-hoc analysis RALES Spironolactone 6 months 261 Spironolactone reduces PICP, PINP, and trial) [36] PIIINP in patients with HFrEF Iraqi et al. (post hoc analysis EPHESUS Eplerenone 6 months 476 Eplerenone reduces PINP and PIIINP in trial) [38] patients with post-MI diastolic HF Ravassa et al. (post hoc analysis ALDO- Spironolactone 12 months 381 Spironolactone reduces PICP and DHF trial) [39] improves diastolic function in patients with HFpEF. Patients with CITP/ MMP-1 < 2.5 show less benefit from treatment Cunningham et al. (post hoc analysis Sacubitril/valsartan 16 weeks 1113 Sacubitril/valsartan reduces TIMP-1, PARAMOUNT trial) [45] sST2, and PIIINP and increases CITP in HFpEF patients compared to vals- artan alone Inflammation modulators RENEWAL trial [47] Etanercept 6 months 2356 Etanercept does not impact on mortality and hospitalizations in patients with HF ATTACH trial [48] Infliximab Discontinued 150 Infliximab increases mortality in patients with HF COLCOT trial [53] Colchicine 22.6 months 4745 Colchicine reduces the risk of ischemic cardiovascular events in patients with MI to 30 days COVERT-MI [55] Colchicine 3 months 192 Colchicine improves the size of the infarcted area after MI and increases the risk of thrombus in LV Abulhul et al. [62] Atorvastatin 6 months 56 Atorvastatin reduces PIIINP levels in patients with systolic HF and normal cholesterol 13 Heart Failure Reviews (2023) 28:555–569 559 Table 1  (continued) Study Therapy Follow-up Included Main evidence patients (n) Chang et al. [63] Atorvastatin 12 weeks 15 Atorvastatin reduces PIIINP and TIMP-1 in hypertensive patients with atherosclerosis Ashton et al. (post-hoc analysis UNI- Rosuvastatin 6 months 32 Rosuvastatin increases PINP and PIIINP VERSE trial) [64] in patients with chronic HF GISSI-HF [66] Rosuvastatin 3.9 years 4574 Rosuvastatin does not improve the prog- nosis of patients with HF CORONA [65] Rosuvastatin 32.8 months 5011 Rosuvastatin does not improve the prog- nosis of patients with systolic HF TGF-β signaling inhibitors AlAnsari et al. [80] Pirfenidone Retrospective study 27 Pirfenidone does not modify echocar- diographic parameters in patients with IPF AlAnsari et al. [81] Pirfenidone Retrospective study 24 Pirfenidone reduces the telesistolic and telediastolic LV volumes in patients with HFpEF and IPF PIROUETTE trial [23] Pirfenidone 52 weeks 94 Pirfenidone slightly reduces ECV, although it does not change diastolic function parameters, in patients with HFpEF MMP inhibitors PREMIER trial [84] PG-116800 90 days 253 PG-116800 does not prevent LV remod- eling nor improve the mortality rate and re-infarction in patients with MI β3-AR modulators BEAT-HF trial [96] Mirabegron 6 months 70 Mirabegron does not improve cardiac function in terms of LVEF in patients with HFrEF volume fraction (CVF) compared to patients treated with (PIIINP) [32]. Similar results were obtained in another study hydrochlorothiazide diuretic (n = 17) [27]. In a study with which included 113 patients with obesity and mild diastolic hypertensive patients treated with losartan (n = 21) and dysfunction [33]. In 44 patients with HF with preserved EF amlodipine (n = 16) with 1-year follow-up, the first group (HFpEF), the administration of eplerenone for 12 months showed reduction of the collagen fraction and the carboxy- reduced PIIINP levels and resulted in a modest improve- terminal pro-peptide of type 1 pro-collagen (PICP) [28]. ment in diastolic function [34]. Similar results emerged in a Another study showed the effectiveness of losartan in study showing that eplerenone reduced the PINP and PICP reducing CVF and increasing left ventricular (LV) com- levels in 44 patients with HFpEF [35]. In the RALES study pliance to 1 year, but only in patients with severe fibrosis (Randomized Aldactone Evaluation Study), conducted in (CVF > 6%, n = 7) [29]. A further study showed attenua- patients with HF with reduced EF (HFrEF), spironolactone tion in the progression of cardiac fibrosis in patients with was associated with reduced mortality and hospitalization, nonobstructive hypertrophic cardiomyopathy treated with as well as with reduction in blood levels of fibrosis bio- losartan [30]. markers and collagen synthesis [36, 37]. In a sub-analysis Aldosterone, whose production is stimulated by Ang of the study EPHESUS (the Eplerenone Post-Acute Myocar- II, also exerts a pro-fibrotic effect in the myocardium by dial Infarction Heart Failure Efficacy and Survival Study), interacting with mineralocorticoid receptors [31]. Aldos- which evaluated the effect of eplerenone in patients with terone receptor antagonists (spironolactone, canrenone, and HF after MI (n = 476), treatment with eplerenone showed eplerenone) showed significant anti-fibrotic effects. In 80 a significant reduction in plasma levels of PINP and PII- patients with metabolic syndrome treated with ACE inhibi- INP (p < 0.007 at 6 months for both), although these were tors (ACEi), treatment with spironolactone for 6 months not associated with prognostic events (mortality and hos- improved diastolic function and decreased levels of PICP pitalization) [38]. Treatment with eplerenone (25 mg/day and amino-terminal pro-peptide of type III pro-collagen up to 50 mg/day, treated n = 3313, placebo n = 3319) was 13 560 Heart Failure Reviews (2023) 28:555–569 also associated with a reduction in the risk of mortality and Colchicine has an important anti-inflammatory action hospitalization for all causes (relative risk (RR): 0.92; 95% because of its effectiveness in inhibiting inflammasome net- CI: [0.86; 0.98]; p = 0.02) after 16 months in treated patients work, various pro-inflammatory cytokines and chemokines with MI complicated by subsequent LV and cardiac dysfunc- [50]. In mouse models of MI, colchicine has been shown tion compared to controls [38]. A sub-study of the ALDO- to be effective in reducing the extent of the infarcted area. DHF trial (The Aldosterone Receptor Blockade in Diastolic The reduction in the extent of fibrosis has been confirmed Heart Failure), which included 381 patients with HFpEF, in a study on rabbit with HF [51, 52]. The COLCOT (COL- identified that treatment with spironolactone reduces PICP CHICINE Cardiovascular Outcomes Trial) study, which levels and improves diastolic function after 12 months of randomized 4745 patients with MI to colchicine or placebo, treatment [39]. revealed a lower risk of ischemic cardiovascular events at Several studies also highlight that the type B natriuretic 30 days from MI in the treated group [53, 54]. The effect peptide (BNP) plays an anti-fibrotic role through the interac- on myocardial fibrosis has not been specifically assessed. tion with its NPR-A and NPR-B receptors and subsequent The recent COVERT-MI study (colchicine for Left ven- activation of cGMP-dependent kinase (PKG) [40]. Although tricular Remodeling Treatment in Acute Myocardial Infarc- the role of the cGMP pathway in fibrosis is not fully under- tion) revealed that patients treated with colchicine after MI stood, several preclinical studies have shown that an increase (n = 101) showed no difference in the size of the infarcted in PKG levels plays an anti-fibrotic effect through negative area at CMR compared to the controls (n = 91) [55]. interference with the TGF-β pathway, which plays a crucial Peroxisome-α proliferator-activated receptor (PPAR-α) role in the activation of fibroblasts [41–44]. The adminis- agonists reduced myocardial fibrosis and improved cardiac tration of sacubitril/valsartan, which represents an associa- function in animal models [56]. On the other hand, exces- tion of ARB and an inhibitor of neprilysin, which is the sive expression of PPAR-α is associated with lipotoxicity, BNP degradation enzyme, has been shown to be effective ventricular dysfunction, and cardiac hypertrophy [57, 58]. in reducing fibrosis. PARAMOUNT (Prospective Compari- Besides reducing cholesterol levels, statins have a pow- son of ARNI With ARB on Management of Heart Failure erful anti-inflammatory and cardioprotective action by with Preserved Ejection Fraction) trial showed that treatment inhibiting the proteins Ras, Rho, and NF-kB, and activat- with sacubitril/valsartan leads to the reduction of plasma ing the PI3K/Akt/Enos pathway [59, 60]. Rosuvastatin has biomarkers of cardiac fibrosis in patients with HFpEF [45]. been shown to be effective in attenuating cardiac fibrosis Sixteen weeks after administration, the treatment group in mouse models of hypertensive heart disease [61]. In a showed a reduction in plasma levels of TIMP-1 (8%; 95% small clinical study, therapy with 40 mg/day of atorvas- CI: [6; 10%]; p < 0.001), soluble suppression of tumorigenic- tatin for 6 months reduced PIIINP levels (4.65 ± 1.86 to ity 2 (sST2) (4%; 95% CI: [1; 7%]; p = 0.002) and PIIINP 4.09 ± 1.25 ng/ml, p < 0.05) in patients with systolic HF (3%; 95% CI: [0; 6%]; p = 0.04), and an increase in carboxy- and normal cholesterol (n = 56) [62]. A study on hyper- terminal telopeptide of type I collagen (CITP) (4%; 95% tensive patients with atherosclerosis (n = 15) treated for CI: [1 to 8%]; p = 0.02), compared to patients treated with the first time with 40 mg/day of atorvastatin for 12 weeks valsartan alone. This additional benefit seems to be caused confirmed the effectiveness of the drug in reducing plasma by the increase in BNP levels [45]. levels of PIIINP (9.5 ± 2.7 to 6.4 ± 1.4 ng/ml, p = 0.012) and TIMP-1 (299 ± 65 to 250 ± 45 ng/ml, p = 0.024) [63]. Inflammation modulators Conflicting results were obtained in a sub-study of the UNIVERSE trial (The rosuvastatin Impact on VEentricu- Tissue damage triggers a phlogistic process that triggers the lar Remodeling cytokineS and neurohormonEs), in which deposition of fibrotic tissue. Tumor necrosis factor α (TNF- an increase in plasma markers of collagen PINP (p = 0.03) α) plays an important role in stimulating cardiac fibrosis and PIIINP (p = 0.001) was observed in patients with [46]. However, the RENEWAL (Randomized etanercept chronic HF (n = 32) treated for 6 months with rosuvasta- Worldwide evaluation) study, which evaluated the effect of tin titrated up to 40 mg/day [64]. In conclusion, the role of the TNF-α antagonist etanercept in patients with HF, showed statins in the treatment of chronic HF is still controversial. no benefit in terms of mortality and hospitalization [47]. Although several retrospective studies have revealed a bet- The ATTACH (anti-TNF Therapy Against Congestive Heart ter prognosis in patients with HF treated with statins, two failure) study was prematurely discontinued due to increased randomized clinical trials, GISSI-HF (Italian Group for the mortality in patients with HF on infliximab, a TNF-α antag- Study of Survival in Heart Failure Failure) and CORONA onist [48]. The later discovery that TNF-1 and TNF-2 recep- (Controlled Rosuvastatin Multinational Trial in Heart tors have opposite effects on cardiac remodeling may partly Failure), reported no prognostic benefit from rosuvastatin explain the disappointing results of TNF-α inhibition [49]. treatment in this class of patients [65, 66]. 13 Heart Failure Reviews (2023) 28:555–569 561 Overall, there is no clear evidence that anti-inflammatory Pirfenidone drugs are effective in reducing cardiac fibrosis. Pirfenidone is an oral anti-fibrotic drug approved for the Anti‑TGF‑β antibodies treatment of IPF [70, 71]. Since pulmonary and cardiac fibrosis share many pathophysiological mechanisms, there Transforming-β growth factor (TGF-β) has a central role is an increasing interest in the application of pirfenidone in the development of cardiac fibrosis. TGF-β achieves its in cardiovascular diseases [72]. The mechanism of action pro-fibrotic effect by the ALK/Smad2/3/Smad4, TAK/p-38/ of pirfenidone remains to be elucidated, but it seems JNK, and NOX4/ROS signaling pathways (Fig. 3) [42, 67]. to reduce the expression of pro-fibrotic factors such as In mouse models of MI and hypertensive heart disease, anti- TGF-β and pro-inflammatory cytokines such as TNF-α, TGF-β and ALK5 antibodies led to reduction of myocar- interleukin (IL)-4, and IL-13 [73]. Pirfenidone also pro- dial fibrosis but not cardiomyocyte hypertrophy. However, motes MMPs expression with subsequent reduction of anti-TGF-β antibody therapy has also been associated with ECM protein accumulation [74]. Pirfenidone could also serious adverse effects, including LV dilation and increased contribute to the modulation of activation and proliferation mortality [68, 69]. The blockade of the TGF-β signaling of T and B cells, thus regulating the secretion of numer- pathway through antibodies therefore seems dangerous, ous pro-inflammatory and pro-fibrotic molecules, such as while less intense inhibition may be more effective. TNF-α and TGF-β [75–77]. Fig. 3  Regulation of TGF-β signaling in cardiac fibrosis. Active vation of Smad-independent pathways, including RhoA and MAPK TGF-β binds to type II and type I receptors, activating downstream signaling, mediates some of the effects of TGF-b in cardiac fibro- Smad-dependent signaling cascades and Smad-independent path- blasts. Endogenous pathways for negative regulation of TGF-b cas- ways. TGF-β binding to the ALK5 type 1 receptor and downstream cades may protect from excessive or unrestrained fibrotic responses. activation of Smad3 signaling induces a matrix-preserving program The inhibitory Smads (Smad6/7), pseudoreceptors such as BAMBI, in cardiac fibroblasts and plays an important role in their activation and soluble endoglin may serve as endogenous inhibitors of TGF-b following cardiac injury. In contrast, the role of ALK1/Smad1/5 sign- signaling, limiting pro-fibrotic responses. Reprinted with permission aling in regulation of fibroblast phenotype is poorly understood. Acti- from Frangogiannis [6] 13 562 Heart Failure Reviews (2023) 28:555–569 In mouse models of hypertension, the administra- Unlike β1- and β2-AR, β3-AR is thought to be resistant tion of pirfenidone has been associated with reduced LV to desensitization because it lacks phosphorylation sites for hypertrophy and increased survival compared to controls GRK kinases [91]. Cardiac expression of β3-AR is physi- [78]. Another study documented that pirfenidone reduces ologically low, but it increases in chronic disease condi- ventricular remodeling and hinders interstitial fibrosis tions [92]. Evidence of the cardioprotective role of β3-ARs induced by Ang II infusion [79]. emerged from the demonstration that mice without β3-ARs Only two retrospective studies evaluated the efficacy of experienced significant cardiac remodeling in response to pirfenidone on cardiac parameters in patients with IPF. In the transverse aortic constriction (TAC) [93]. β3-AR agonists first, no association between administration of pirfenidone (BRLs) have proven effective in counteracting cardiac fibro- and change in echocardiographic parameters was found [80]. sis in several preclinical models of cardiovascular disease. In In the second, pirfenidone was associated with a reduction murine models of HFpEF induced by infusion of Ang II, the in end-systolic and end-diastolic LV volumes, but without administration of BRLs has shown beneficial effects associ- significant changes in ventricular function [81]. The PIR- ated with attenuation of cardiac fibrosis, including improve- OUETTE phase II study included 94 patients with HFpEF ment of myocardial stiffness and reduction of pulmonary and extended fibrosis, defined as 27% ECV [23]. After a congestion [94]. In mice knockout for neuronal nitric oxide 52-week follow-up, an absolute reduction of 0.7% in the synthase (nNOS) undergoing TAC, the administration of ECV in pirfenidone-treated group was found compared to BRLs for 3 weeks revealed attenuation of ventricular dila- an increase of 0.5% in placebo-treated controls (p = 0.009). tion, systolic dysfunction, and partial reduction of cardiac This very limited effect on the ECV was not associated with hypertrophy [95]. However, the first randomized controlled significant changes in diastolic function parameters [23]. trial that evaluated the effectiveness of the β3-AR mira- begron agonist in the HFrEF, namely, the BEAT-HF trial MMP inhibitors (Baroreflex Activation Therapy for Heart Failure), showed no improvement in cardiac function in terms of EF variation Although MMP inhibitors (MMPi) have been shown to over a period of 6 months [96]. attenuate cardiac fibrosis and remodeling in experimental models, these results have not achieved the expected clinical results [82, 83]. In the PREMIER (Prevention of Myocardial Anti‑fibrotic therapy with CAR‑T cells Infarction Early Remodeling) study, in which the effect of oral administration of PG-116800 in 253 patients with recent Ex vivo engineered CAR‑T cell therapy MI was evaluated, the drug has not shown any beneficial effect in preventing LV remodeling or improving the mortal- CARs are engineered receptors that function to redirect lym- ity rate or re-infarction at 90 days after MI [84]. phocytes, most commonly T cells, to recognize and eliminate cells expressing a specific target antigen. This interaction Adrenergic receptor system modulators occurs in a specific CAR domain called “antigen binding domain” and allows endogenous activation of T cells, with Pharmacological blockade of β1 adrenergic receptors subsequent elimination of target cells [97, 98]. CAR-T cell (β1-ARs) is a fundamental therapy for the treatment of HF therapy represents a revolutionary therapeutic approach with and for the prevention of structural remodeling, prevent- significant and lasting clinical benefits already demonstrated ing and attenuating progressive dilation of LV and cardiac in hematological diseases, such as large B-cell lymphoma hypertrophy [85–87]. In contrast, the signaling pathway or acute lymphoblastic leukemia (ALL) [99]. CAR-T cell of β2-AR plays anti-fibrotic effects. However, while the therapy was approved by the Food and Drug Administration acute effect of β2-AR activation inhibits collagen synthesis, (FDA) in 2017 for the treatment of ALL [100]. myofibroblasts extracted and isolated from patients with HF Aghajanian et al. firstly investigated the effectiveness appear to be resistant to β2-AR agonists, possibly due to the of ex vivo engineered T cells for the selective elimination increased activity of GPCR-2 kinase (GRK2) [88]. In fact, of myofibroblasts expressing the fibroblast activation pro- numerous studies on mouse models have highlighted the tein (FAP) on the membrane surface, in a mouse model of pathological role of GRK2 in HF. GRK2 became an attrac- fibrosis induced by infusion of Ang II and phenylephrine tive pharmacological target after the discovery that its inhi- [101]. The authors identified FAP as the best marker of bition is associated with significant protection against myo- cardiac myofibroblasts since peptide expression is almost cardial fibrosis in HF animal models [89]. In a pig model of exclusive to this cell population. The use of CAR-T cells post-MI HF, adeno-associated virus gene therapy allowed directed against FAP resulted in the reduction of fibrosis the expression of a GRK2 inhibitor peptide (βARKct), dem- in all seven treated mice, and an almost total elimination onstrating long-term improvement of heart function [90]. in 5/7, as well as the maintenance of normal systolic and 13 Heart Failure Reviews (2023) 28:555–569 563 diastolic function [101]. A confirmation of the selectivity failure, equal to 20–50% [104, 105]. Potential toxic- of CAR-T cells is provided by the persistence of perivas- ity associated with CAR-T cell therapy has stimulated cular fibrosis after treatment since perivascular fibroblasts the search for alternative approaches, such as the use of do not expose FAP on the membrane surface and therefore CAR-Natural Killer (NK) cells, and safer cell program- do not represent a target for CAR-T FAP cells. In addi- ming methods. tion, the administration of engineered T cells in mice with fibrosis was shown to be safe and poorly cardiotoxic, with Use of CAR‑T cells for the treatment of myocardial a very mild inflammatory response [101]. fibrosis in vivo CAR-T cells are usually produced ex vivo by transduc- tion using a retrovirus or lentivirus containing a DNA or Rurik et al. developed a strategy to obtain anti-fibrotic RNA coding for a genetically modified CAR protein, in transient CAR-T cells using a system of lipid nanoparticles autologous T cells taken from the patient. Once modified, (LNPs) that enclose modified mRNA [24]. The introduction the T cells are expanded and infused into the patient with of mRNA into T cells to transform them into CAR-T cells prior depletion of naïve T cells (Fig. 4) [102]. This method had already been performed through electroporation method, is effective but long and expensive. A further limitation of but exclusively ex vivo [106]. To avoid the need for extrac- ex vivo engineered CAR-T cells is their persistent activa- tion and reinfusion of T cells, the authors have developed an tion after infusion [103]. approach to achieve in vivo differentiation of CAR-T cells The most common reported adverse effect is the syn- by infusion of LNP containing modified mRNA. The use of drome of release of pro-inflammatory cytokines by LNP-mRNA systems has been very successful in the pro- activated CAR-T cells and other immune cells, with an duction of vaccines against COVID-19 and in other clinical incidence of mild complications of 70–90% and severe contexts since LNPs can be coated with antibodies directed complications, such as cardiogenic shock and multiorgan against a specific target cell, such as T cells, to which they Fig. 4  Ex vivo production of CAR-T cells. Autologous T cells are extracted from the patient, then they are engineered in the laboratory to obtain CAR expression and the differen- tiation of T lymphocytes into CAR-T cells, which will then be amplified and re-infused into the patient with prior lym- phodepletion. Reprinted with permission from https://​www.​ cancer.​gov/​about-​cancer/​treat​ ment/​resea​rch/​car-t-​cells 13 564 Heart Failure Reviews (2023) 28:555–569 Fig. 5  In vivo production of CAR-T cells using CD5/LNP-FAPCAR. blasts, allows to obtain transients CAR-T cells in vivo that specifically Administration of LNP coated with anti-CD5 antibodies and contain- eliminate the pro-fibrotic cells from the injured myocardium. CAR, ing mRNA coding for the FAPCAR membrane receptor, which selec- chimeric antigen receptor; FAP, fibroblast activation protein; LNP, tively recognizes the FAP protein expressed by cardiac myofibro- lipid nanoparticle. Reprinted with permission from Rurik et al. [24] release the mRNA [107]. The authors managed to produce Histological analysis showed a significant reduction in the CAR-T cells less stable and durable than that normally pro- ECM fraction, with regression of interstitial fibrosis, so that duced ex vivo, avoiding the problem of their indefinite per- 5 of the 12 samples of treated mice were indistinguishable manence and activation. from sham animals [24]. Ex vivo production of CAR-T cells consists in the trans- Overall, the use of in vivo engineered CAR-T cells via duction with viral vector and the integration of a fragment LNP-mRNA and directed against FAP results in the elimina- of nucleic acid coding for the engineered CAR protein in tion of cardiac myofibroblasts, in the reduction of myocar- the genome of the target cell. On contrast, the in vivo infu- dial fibrosis and improvement of cardiac geometry and func- sion through LNP-mRNA system leads to endocytosis of tion, potentially exceeding the limits of ex vivo engineering the LNP, resulting in the degradation and thus the release of of CAR-T cells. mRNA, which is incapable of genomic integration and has a limited half-life, into the T cells’ cytoplasm [24] (Fig. 5). Feasibility of CAR‑T cell therapy in humans Rurik et al. first designed a modified mRNA coding for a CAR receptor directed against the FAP protein (FAP- The infusion of CAR-T cells is emerging as a good approach CAR), expressed by cardiac fibroblasts activated by tissue to counteract cancer development and the number of poten- damage, and encapsulated it within an anti-CD5 antibody- tial targets suitable for CAR-T cell therapy is growing rap- coated LNP (CD5/LNP-FAPCAR). CD5 is a membrane gly- idly. The heart and the immune system are highly cross- coprotein physiologically expressed by T cells [108]. The linked and accumulating data suggest that the modulation authors subsequently tested the effectiveness of the CD5/ of immunological cells may provide beneficial effects in LNP-FAPCAR system in transforming a culture of T cells many cardiovascular diseases, such as cardiac fibrosis, cor- into CAR-T cells, and their ability to eliminate target cells onary atherosclerosis, and HF [110, 111]. The successful with FAP expression in vitro. The positive results encour- application of CAR-T cells to cancer treatment could be thus aged the translation of the method in vivo. Mice injected translated to other fields, making the CAR-T cells immuno- with Ang II and phenylephrine for 28 days then received therapy a potential approach against cardiovascular diseases. intravenous administration of CD5/LNP-FAPCAR [109]. However, there are still important limitations to CAR-T At 48 h after the injection, the authors detected a signifi- cell therapy that need to be solved. One of the most common cant expression of FAPCAR in a portion (17.5–24.7%) of limitations of CAR-T cell therapy in oncological disease is T cells (FAPCAR + -T), in the treated group [24]. Treated the development of the so-called antigen escape, which is the mice showed improved size, systolic and diastolic function, acquired tissue resistance to single antigen targeting. The cells and LV mass compared to untreated mice [24] (Fig. 6). of a significant portion of patients treated with CAR-T cells 13 Heart Failure Reviews (2023) 28:555–569 565 Fig. 6  In vivo engineered CAR-T cells against FAP improve cardiac nal section of uninjured mice (n = 8, 3 weeks after saline infusion function after myocardial damage. Adult wild-type mice C57BL/6 pump implantation + 1-week saline injection), damaged control mice received a continuous infusion with saline or Ang II + PE via mini- (n = 11, Ang II + PE + saline), and damaged treated mice (n = 12, osmotic pump implanted for 28 days. After a week of heart dam- Ang II + PE + CD5/LNP-FAPCAR). The quantification of fibrosis age due to pressure overload, mice received a single dose of 10 mg is expressed as a percentage of the observed ventricle. The data are of CD5/LNP-FAPCAR. Mice were analyzed 2 weeks after treat- expressed as average ± standard error. The p values shown derive ment. Telediastolic (A) and telesystolic (B) volume measurement from Tukey’s post hoc test after one-way ANOVA (p < 0.05). Ang of LV. Left Ventricular Mass Index (LVMI) (C), diastolic function II, angiotensin II; EF, ejection fraction; PE, phenylephrine. Modified (E/e ratio) (D), EF (E), and global longitudinal strain (F) estima- with permission from Rurik et al. [24] tion. Picrosirius red staining (G) highlights collagen (pink) in coro- may partly or completely lose the target antigen expression Conclusion over time, thus eluding the CAR-T cells [112]. Moreover, CAR-T cell therapy is highly associated with life-threatening The pathophysiological heterogeneity of myocardial fibrosis toxicities and cardiovascular events such as symptomatic heart and the complexity of tissue fibroblasts’ response to induced failure, acute coronary syndrome, ischemic stroke, and de novo damage complicate the development of anti-fibrotic strat- cardiac arrhythmia [113]. The toxic effects of CAR-T cells egies in cardiovascular diseases. Some drugs have proven on cardiovascular system need to be further elucidated [113, effective in reducing further deposition of ECM in the myo- 114]. In conclusion, new strategies are emerging, and they cardium, such as RAAS inhibitors. 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