Elimination or neutralization of endogenous high-molecular-weight FGF2 mitigates doxorubicin-induced cardiotoxicity.

Cardiac fibroblast growth factor 2 (FGF2) exerts multiple paracrine activities related to cardiac response to injury. Endogenous FGF2 is composed of a mixture of 70% high- and 30% low-molecular-weight isoforms (Hi-FGF2 and Lo-FGF2, respectivley); although exogenously added Lo-FGF2 is cardioprotective, the roles of endogenous Hi-FGF2 or Lo-FGF2 have not been well defined. Therefore, we investigated the effect of elimination of Hi-FGF2 expression on susceptibility to acute cardiac damage in vivo caused by an injection of the genotoxic drug doxorubicin (Dox). Mice genetically depleted of endogenous Hi-FGF2 and expressing only Lo-FGF2 [FGF2(Lo) mice] were protected from the Dox-induced decline in ejection fraction displayed by their wild-type FGF2 [FGF2(WT)] mouse counterparts, regardless of sex, as assessed by echocardiography for up to 10 days post-Dox treatment. Because cardiac FGF2 is produced mainly by nonmyocytes, we next addressed potential contribution of fibroblast-produced FGF2 on myocyte vulnerability to Dox. In cocultures of neonatal rat cardiomyocytes (r-cardiomyocytes) with mouse fibroblasts from FGF2(WT) or FGF2(Lo) mice, only the FGF2(Lo)-fibroblast cocultures protected r-cardiomyocytes from Dox-induced mitochondrial and cellular damage. When r-cardiomyocytes were cocultured with or exposed to conditioned medium from human fibroblasts, neutralizing antibodies for human Hi-FGF-2, but not total FGF2, mitigated Dox-induced injury of cardiomyocytes. We conclude that endogenous Hi-FGF2 reduces cardioprotection by endogenous Lo-FGF2. Antibody-based neutralization of endogenous Hi-FGF2 may offer a prophylactic treatment against agents causing acute cardiac damage. NEW & NOTEWORTHY Cardiomyocytes, in vivo and in vitro, were protected from the deleterious effects of the anticancer drug doxorubicin by the genetic elimination or antibody-based neutralization of endogenous paracrine high-molecular-weight fibroblast growth factor 2 isoforms. These findings have a translational potential for mitigating doxorubicin-induced cardiac damage in patients with cancer by an antibody-based treatment.

High molecular weight fibroblast growth factor-2 in the human heart is a potential target for prevention of cardiac remodeling.

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein synthesized as high (Hi-) and low (Lo-) molecular weight isoforms. Studies using rodent models showed that Hi- and Lo-FGF-2 exert distinct biological activities: after myocardial infarction, rat Lo-FGF-2, but not Hi-FGF-2, promoted sustained cardioprotection and angiogenesis, while Hi-FGF-2, but not Lo-FGF-2, promoted myocardial hypertrophy and reduced contractile function. Because there is no information regarding Hi-FGF-2 in human myocardium, we undertook to investigate expression, regulation, secretion and potential tissue remodeling-associated activities of human cardiac (atrial) Hi-FGF-2. Human patient-derived atrial tissue extracts, as well as pericardial fluid, contained Hi-FGF-2 isoforms, comprising, respectively, 53%(±20 SD) and 68% (±25 SD) of total FGF-2, assessed by western blotting. Human atrial tissue-derived primary myofibroblasts (hMFs) expressed and secreted predominantly Hi-FGF-2, at about 80% of total. Angiotensin II (Ang II) up-regulated Hi-FGF-2 in hMFs, via activation of both type 1 and type 2 Ang II receptors; the ERK pathway; and matrix metalloprotease-2. Treatment of hMFs with neutralizing antibodies selective for human Hi-FGF-2 (neu-AbHi-FGF-2) reduced accumulation of proteins associated with fibroblast-to-myofibroblast conversion and fibrosis, including α-smooth muscle actin, extra-domain A fibronectin, and procollagen. Stimulation of hMFs with recombinant human Hi-FGF-2 was significantly more potent than Lo-FGF-2 in upregulating inflammation-associated proteins such as pro-interleukin-1ß and plasminogen-activator-inhibitor-1. Culture media conditioned by hMFs promoted cardiomyocyte hypertrophy, an effect that was prevented by neu-AbHi-FGF-2 in vitro. In conclusion, we have documented that Hi-FGF-2 represents a substantial fraction of FGF-2 in human cardiac (atrial) tissue and in pericardial fluid, and have shown that human Hi-FGF-2, unlike Lo-FGF-2, promotes deleterious (pro-fibrotic, pro-inflammatory, and pro-hypertrophic) responses in vitro. Selective targeting of Hi-FGF-2 production may, therefore, reduce pathological remodelling in the human heart.

Preferential accumulation and export of high molecular weight FGF-2 by rat cardiac non-myocytes.

AIMS: fibroblast growth factor-2 (FGF-2), implicated in paracrine induction of cardiac hypertrophy, is translated as high molecular weight (Hi-FGF-2) and low molecular weight (Lo-FGF-2) isoforms. Paracrine activities are assigned to Lo-FGF-2, whereas Hi-FGF-2 is presumed to have nuclear functions. In this work, we re-examined the latter presumption by asking whether: cardiac non-myocytes (CNMs) accumulate and export Hi-FGF-2 in response to pro-hypertrophic [angiotensin II (Ang II)] stimuli; an unconventional secretory pathway requiring activated caspase-1 affects Hi-FGF2 export; and secreted Hi-FGF-2 is pro-hypertrophic. METHODS AND RESULTS: using neonatal rat heart-derived cultures and immunoblotting, we show that CNMs accumulated over 90% Hi-FGF-2, at levels at least five-fold higher than cardiomyocytes (CMs). Pro-hypertrophic agents (Ang II, endothelin-1, and isoproterenol) up-regulated CNM-associated Hi-FGF-2. The Ang II effect was mediated by Ang II receptor-1 but not Ang II receptor-2 as it was blocked by losartan but not PD123319. CNM-derived Hi-FGF-2 was detected in two extracellular pools: in conditioned medium from Ang II-stimulated CNMs and in association with the cell surface/matrix, eluted with a gentle 2 M NaCl wash of the cell monolayer. Conditioned medium from Ang II-treated CNMs increased neonatal CM size, an effect prevented by anti-FGF-2-neutralizing antibodies. The caspase-1 inhibitor YVAD prevented the Ang II-induced release of Hi-FGF-2 to both extracellular pools. CONCLUSION: CNMs are major producers of Hi-FGF-2, up-regulated by hypertrophic stimuli and exported to the extracellular environment by a mechanism requiring caspase-1 activity, suggesting a link to the innate immune response. Hi-FGF-2 is likely to promote paracrine induction of myocyte hypertrophy in vivo.

A high-lipid diet potentiates left ventricular dysfunction in nitric oxide synthase 3-deficient mice after chronic pressure overload.

A high-lipid diet (HLD) may lead to adverse left ventricular (LV) remodeling and endothelial dysfunction in conditions of hemodynamic stress. Although congenital absence of nitric oxide synthase 3 (NOS3) leads to adverse LV remodeling after transverse aortic constriction (TAC), the effects of a HLD in this state remains unknown. Wild-type (WT) and NOS3 knockout mice (NOS3(-/-)) were randomized into the following 4 groups: 1) WT + low-lipid diet (LLD) (10% of energy); 2) WT + HLD (60% of energy); 3) NOS3(-/-) + LLD; and 4) NOS3(-/-) + HLD for a total of 12 wk. After 1 wk of randomization, TAC was performed on all groups. Serial echocardiography revealed a decrease in LV ejection fraction (LVEF) in WT and NOS3(-/-) mice fed the HLD compared with those fed the LLD diet at 12 wk post-TAC. Mice fed the NOS3(-/-) + HLD diet had a lower LVEF compared with mice in the other 3 groups (P < 0.05). There was greater myocyte hypertrophy, interstitial fibrosis, and percentage change in plasma cholesterol concentrations in the NOS3(-/-) + HLD group 12 wk post-TAC compared with the other 3 groups. Although high molecular weight fibroblast growth factor-2, a marker of cardiac hypertrophy, was more upregulated in the NOS3(-/-) + HLD group than in the other groups, markers of the renin-angiotensin system did not differ among them. A HLD potentiates LV dysfunction in NOS3(-/-) mice in a chronic pressure overload state.

Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts.

In fibrosing hearts, myofibroblasts are associated with cardiac extracellular matrix remodeling. Expression of key genes in the transition of cardiac fibroblast to myofibroblast phenotype in post-myocardial infarction heart and in vitro has not been well addressed. Contractile, focal adhesion-associated, receptor proteins, fibroblast growth factor-2 (FGF-2) expression, and motility were compared to assess phenotype in adult and neonatal rat cardiac fibroblasts and myofibroblasts. Neonatal and adult fibroblasts undergo phenotypic transition to myofibroblastic cells, marked by increased alpha-smooth muscle actin (alphaSMA), smooth muscle myosin heavy chain (SMemb), extra domain-A (ED-A) fibronectin, paxillin, tensin, FGF-2, and TbetaRII receptor. Elevated ED-A fibronectin confirmed fibroblast to supermature myofibroblastic phenotype transition. Presence of myofibroblasts in vivo was noted in sections of healed infarct scar after myocardial infarction, and their expression is similar to that in culture. Thus, cultured neonatal and adult cardiac fibroblasts transition to myofibroblasts in vitro and share expression profiles of cardiac myofibroblasts in vivo. Reduced motility with in vitro passage reflects enhanced production of focal adhesions.

Fibroblast growth factor-2 and cardioprotection.

Boosting myocardial resistance to acute as well as chronic ischemic damage would ameliorate the detrimental effects of numerous cardiac pathologies and reduce the probability of transition to heart failure. Experimental cardiology has pointed to ischemic and pharmacological pre- as well as post-conditioning as potent acute cardioprotective manipulations. Additional exciting experimental strategies include the induction of true regenerative and/or angiogenic responses to the damaged heart, resulting in sustained structural and functional beneficial effects. Fibroblast growth factor-2 (FGF-2), an endogenous multifunctional protein with strong affinity for the extracellular matrix and basal lamina and well-documented paracrine, autocrine and intracellular modes of action, has been shown over the years to exert acute and direct pro-survival effects, irrespectively of whether it is administered before, during or after an ischemic insult to the heart. FGF-2 is also a potent angiogenic protein and a crucial agent for the proliferation, expansion, and survival of several cell types including those with stem cell properties. Human clinical trials have pointed to a good safety record for this protein. In this review, we will present a case for the low molecular weight isoform of fibroblast growth factor-2 (lo-FGF-2) as a very promising therapeutic agent to achieve powerful acute as well as sustained benefits for the heart, due to its cytoprotective and regenerative properties.