Genetic ablation of fibroblast activation protein alpha attenuates left ventricular dilation after myocardial infarction

Introduction Regulating excessive activation of fibroblasts may be a promising target to optimize extracellular matrix deposition and myocardial stiffness. Fibroblast activation protein alpha (FAP) is upregulated in activated fibroblasts after myocardial infarction (MI), and alters fibroblast migration in vitro. We hypothesized that FAP depletion may have a protective effect on left ventricular (LV) remodeling after MI. Materials and methods We used the model of chronic MI in homozygous FAP deficient mice (FAP-KO, n = 51) and wild type mice (WT, n = 55) to analyze wound healing by monocyte and myofibroblast infiltration. Heart function and remodeling was studied by echocardiography, morphometric analyses including capillary density and myocyte size, collagen content and in vivo cell-proliferation. In non-operated healthy mice up to 6 months of age, morphometric analyses and collagen content was assessed (WT n = 10, FAP-KO n = 19). Results Healthy FAP-deficient mice did not show changes in LV structure or differences in collagen content or cardiac morphology. Infarct size, survival and cardiac function were not different between FAP-KO and wildtype mice. FAP-KO animals showed less LV-dilation and a thicker scar, accompanied by a trend towards lower collagen content. Wound healing, assessed by infiltration with inflammatory cells and myofibroblasts were not different between groups. Conclusion We show that genetic ablation of FAP does not impair cardiac wound healing, and attenuates LV dilation after MI in mice. FAP seems dispensable for normal cardiac function and homeostasis.


Results
Healthy FAP-deficient mice did not show changes in LV structure or differences in collagen content or cardiac morphology. Infarct size, survival and cardiac function were not different between FAP-KO and wildtype mice. FAP-KO animals showed less LV-dilation and a thicker scar, accompanied by a trend towards lower collagen content. Wound healing, assessed by infiltration with inflammatory cells and myofibroblasts were not different between groups.

Conclusion
We show that genetic ablation of FAP does not impair cardiac wound healing, and attenuates LV dilation after MI in mice. FAP seems dispensable for normal cardiac function and homeostasis.

Introduction
The myocardial extracellular matrix (ECM) is a critical component in normal and pathophysiological conditions of the heart, and is mainly regulated by cardiac fibroblasts [1]. During early ventricular remodeling after myocardial infarction (MI) the invasion and activity of myofibroblasts is critical for wound healing and scar development [2,3]. However, elevated deposition of ECM by fibroblasts leads to cardiac dysfunction in late remodeling [4]. Protecting the left ventricle (LV) from detrimental remodeling after MI is still a challenge. Even if the current medical treatment options after MI including RAAS inhibitors, mineralocorticoid antagonists and beta blockers show beneficial effects, there is still no specific antifibrotic treatment option for chronic ventricular remodeling [4]. The dipeptidyl-peptidase Fibroblast activation protein α (FAP) is a serine protease expressed by activated fibroblasts after MI in animals and humans [5,6]. FAP is highly expressed in activated fibroblasts by a TGFβ-driven mechanism after MI in rats, and promotes fibroblast migration and exerts gelatinolytic activity in vitro [5]. FAP pos activated fibroblasts are present in hearts of patients with chronic ischemic cardiomyopathy, demonstrating persistent fibrotic activity in these patients [5]. FAP was also detected in human atherosclerotic plaques and associated with plaque progression and fibrous cap thinning [7], whereas deletion of FAP decreased atherosclerotic plaque formation in a mouse model [8]. Increased expression of FAP was also found in pathological fibrotic diseases like idiopathic pulmonary fibrosis [9], liver cirrhosis [10] and keloids [11] as well as in stromal soft tissue of several kinds of cancer [12][13][14]. In healthy hearts and other tissues the expression of FAP is absent or very low [12,15].
A first successful attempt has been described to reduce cardiac fibrosis by targeting FAPexpressing fibroblasts in rodents using antigen-specific CD8 pos T cells in angiotensin II/phenylephrine induced myocardial fibrosis [16]. Because therapies targeting FAP pos myofibroblasts will also alter myocardial FAP levels, it is important to understand the function and pathophysiological significance of FAP deficiency in normal healthy hearts and post-MI in vivo. Since FAP is upregulated in fibrotic diseases and especially after MI and alters fibroblast migration, we hypothesized that FAP depletion may have a protective effect on LV remodeling after MI.

Materials and methods
Additional materials and methods are presented as Supplementary Online Material.

Experimental myocardial infarction in mice
Myocardial infarction (MI) was induced in female homozygous FAP-deficient mice (FAP-KO, n = 51) on a C57BL/6NCrl background [17], generated by Niedermeyer et al. [18], or wildtype C57BL/6NCrl mice (n = 55) aged 12 weeks as described previously [19,20]. Briefly, under 1.5-2% isoflurane anesthesia (induction with 5% isoflurane), the thorax was opened and the proximal left anterior descending coronary artery was occluded using a 5-0 suture. Animals were kept warm with a heating pad. Depth of anesthesia was tested using the pedal withdrawal reflex. Analgesia was maintained using buprenorphine (0.05 mg/kg BW i.p.). Before and after surgery, animals were housed in the animal facility and monitored daily for activity and signs of pain. Surviving animals were euthanized by cardiac arrest using intracardiac injection of saturated potassium chloride solution and hearts removed at 7 days and 4 weeks after MI. An additional group of mice was studied without surgery for six months to assess physiological changes in animals (WT n = 10, FAP-KO n = 19). The hearts were removed for anatomical, histological and western blot analyses and fixed with paraformaldehyde or frozen. Short term survival analysis was performed in a subgroup of 56 operated animals (WT operated n = 26, surviving n = 12; FAP-KO n = 30, surviving n = 12). Analysis for ventricular rupture was performed by macroscopic inspection in a subgroup of WT (n = 11) and FAP KO (n = 13) operated animals. Animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the local government (Regierung von Unterfranken permission number K 55.2-2531.01-64/09).

Echocardiographic analysis
We performed serial transthoracic echocardiography at days 1, 14 and 28 after MI by an experienced technician as described previously by Vogel et al. [21]. Echocardiography was performed under isoflurane anesthesia and spontaneous respiration. The endocardial borders were traced at end-systole and end-diastole with the help of a prototype analysis off-line system (NICE; Toshiba Medical System, Netherlands). Parameters were measured at the mid-papillary and apical muscle level in B-Mode images.

Immunohistochemistry of mouse myocardial tissue
Frozen or formalin-fixed paraffin-embedded sections from mouse hearts were stained with antibodies against CD68, α smooth muscle actin (SMA) and CD31 and quantified as described in S1 Material. At 28 days after MI, hearts were analyzed for the number of CD31 pos capillaries. For analysis of myocyte size, H&E stained formalin fixed paraffin embedded sections were imaged at 20x magnification, and areas of cross sectioned myocytes were analyzed in the intact myocardium using Image Pro Plus software (Media Cybernetics, Bethesda, USA).

Analysis of cell proliferation in vivo
For detection of myocardial cell proliferation in vivo, MI was induced in female WT and FAP-KO mice at 12 weeks of age (n = 4 each). BrdU (Roche), 50 mg per kg body weight, was injected twice a day every day before sacrifice on day 14. For immunofluorescent staining of BrdU, formalin fixed, paraffin embedded tissues were sectioned at 4 μm, and heat induced antigen retrieval was performed using Histosafe Enhancer (Linaris, Germany). After a blocking-step using 10% donkey serum in PBS, sections were sequentially incubated with primary antibodies against BrdU (5-Bromo-2deoxy-uridine Labeling and detection kit, Roche) and detected by fluorescent secondary antibodies (Jackson ImmunoResearch). Nuclear DNA was labeled using DAPI (Invitrogen). Images were obtained at 20x with a Nikon NiE microscope, quantitative analysis of BrdU-positive and BrdU-negative nuclei was performed in the scar and the surviving free wall adjacent to the scar using Image Pro Plus software (Media Cybernetics, Bethesda, USA), and proliferation index was calculated. Image processing with Photoshop (Adobe) included changes in brightness, contrast and tonal range, and was applied equally across the entire image.

Analysis of collagen content after MI and in healthy mice
Collagen content in the intact myocardium was analyzed 28d after MI by analyzing picrosirius red stained tissues sections. In healthy mice 6 months of age, collagen content was measured by use of hydroxyproline assay.
by Fisher's exact test. A value of P less than 0.05 was considered statistically significant. Statistical analysis was performed with Prism 5 (GraphPad).

FAP deficiency does not impact LV morphology and fibrosis in healthy mice
We performed histological analyses in nonoperated healthy mice up to the age of six months. No differences between FAP-KO mice and WT mice in body weight, heart weight, LV morphology were observed (S1 Fig). Of note, collagen content, as measured by hydroxyproline assay, was similar in both groups indicating unaltered collagen homeostasis in healthy mice up to 6 months of age. Moreover, isolated fibroblasts from hearts of WT and FAP-KO mice showed no overt difference in phenotype and growth properties (S2 Fig).

LV dilation is reduced in FAP deficient animals after MI
We used the established model of chronic occlusion of the left coronary artery to induce large MI. Infarct size did not differ significantly between the FAP-KO group and WT group 7 and 28 days after MI (Fig 1A). Postoperative survival rate was similar in FAP-KO and WT mice two weeks after MI (WT: 50%, FAP-KO: 45%, n.s., Fig 1B). No difference in number of ventricular ruptures between FAP-KO and WT mice was observed in a subgroup of infarcted mice: Ventricular rupture was assumed in 5 out of 11 (45%) infarcted WT animals, and in 5 out of 13 (39%) infarcted FAP-KO animals (n.s.). FAP deficiency in FAP-KO animals was confirmed by western blot in infarcted hearts and in isolated cardiac fibroblasts from normal healthy hearts (S2A and S2B Fig).
We performed echocardiographic analyses to examine functional effects of FAP deficiency at days 1, 14 and 28 after MI (Fig 2; S1 Table). Corresponding to the reduced infarct expansion index in FAP-KO animals, end-diastolic area at papillary muscle level was decreased at 14 days (-21%, p<0.05) and 28 days (-17%, p<0.05) after MI in FAP-KO animals as compared to WT animals. Furthermore, end-systolic area at the papillary muscle level was also decreased 28 days after MI in FAP-KO animals (-21%, p<0.05). In agreement with the previous findings a trend towards reduced end-systolic and end-diastolic LV area was also detected when measured at the LV apical levels (S1 Table). LV systolic function as measured by fractional shortening was not different between groups at both time points.
Together, these results demonstrate improved LV remodeling by reduced LV dilation in FAP-KO animals at 28 days after MI.

Monocyte and fibroblast infiltration are not altered in FAP deficient mice after MI
To understand possible mechanisms responsible for improved LV remodeling in FAP-KO mice, we analyzed the myocardium by immunohistochemistry at 7 days after MI. In both groups, infarcted myocardium was infiltrated with SMA pos myofibroblasts and CD68 pos monocytes at 7d after MI, and no difference in expression of both markers was apparent between groups (Fig 3A and 3B). Additionally, total cell density and cell proliferation in intact

PLOS ONE
FAP-KO attenuates LV dilation after MI or infarcted myocardium was not different between groups at 14d after MI, respectively ( Fig  3C), indicating integrity of the cellular wound healing response after MI in FAP-KO mice.
Next, we analyzed the intact myocardium by immunohistochemistry at 28 days after MI to evaluate possible effects of FAP deficiency on myocytes, capillaries and collagen content. There were no differences in myocyte cross sectional area as well as capillary density in FAP-KO and WT mice (Fig 4). Collagen content, as measured by picrosirius red staining, showed a modest trend towards less collagen deposition in the FAP-KO group (n.s.).
Together, the morphometric results indicate unaltered inflammatory and fibroblast infiltration at the early wound healing phase, and a slight trend towards less collagen deposition in the chronic remodeling phase after MI in FAP-KO animals.

Discussion
The serine protease FAP is upregulated after MI and primarily identifies activated myocardial fibroblasts [5,6], but so far no studies have analyzed the physiological role of FAP in the heart in vivo. In this study, we show that genetic ablation of FAP does not alter cardiac wound healing but attenuates LV dilation after MI in mice.

FAP deficiency attenuates LV dilation without affecting collagen content after MI
We analyzed the role of FAP on LV geometry and scar morphology after MI and show that the minimal and average scar thickness was greater in FAP deficient animals as compared to wildtype animals, thus attenuating LV dilation after 28 days. This data was also supported by echocardiography showing less LV dilation in FAP deficient animals. At the same time, animals did not show differences in signs of heart failure as body weight, heart and lung weights were not different in both groups.
Adverse cardiac remodeling with LV enlargement determines clinical impairment and mortality [22]. Therefore, improvement of cardiac remodeling is one of the main aims of current heart failure therapy [4]. LV morphology and dilation is dependent on collagen accumulation and structure, and balanced matrix degradation and production is a hallmark of post-MI wound healing. Matrix degradation is primarily performed by enzymes such as matrix metalloproteinases (MMP). In fact, inhibition of MMP activity has been studied extensively and shown to improve myocardial remodeling [23]. Genetic deletion of MMP-9 improved LV remodeling after MI [24]. Instead, cardiac overexpression of membrane type-1 matrix metalloproteinase (MT1-MMP) resulted in reduced LV function and increased fibrosis after MI [25]. In the study by Ducharme et al., LV dilation was accompanied by reduced collagen content and reduced inflammatory cell infiltration in MMP9-deficient mice after MI [24]. In comparison to these studies, we found only mild improvement in LV geometry in FAP-KO animals without difference in LV function, which might be a result of the only slightly reduced collagen content in the FAP-KO animals after MI, indicating that FAP has only minor effects on collagen homeostasis in the heart in vivo within the first 4 weeks after MI.
FAP is induced by TGFβ and TGFβ is one of the main profibrotic cytokines in the heart, highly upregulated after MI and necessary for improved wound healing and remodeling [5,26,27]. However, the reason why we did not find significant differences in collagen deposition between groups remains unclear. In this regard, a study using a TGFβ-overexpression model of chronic pulmonary fibrosis in mice also demonstrated only little effects of FAP deficiency on pulmonary fibrotic response [28]. This suggests in line with our results that TGFβ-induced tissue fibrosis is not mediated nor altered by FAP. Moreover, we demonstrated that 6 months old animals did not show any difference in LV morphology, indicating that FAP is not essential in normal myocardial homeostasis. This is further supported by data from Niedermayer et al. describing no developmental defects and normal heart morphology in FAP-deficient mice [18].

Wound healing is not altered in FAP deficient animals
The initial wound healing phase after MI is critical for myocardial healing and paves the way for infarct repair [29], and depletion of monocytes/macrophages after MI leads to severely compromised extracellular matrix remodeling and increased infarct expansion [30]. In our study we found inflammatory cell infiltration, myofibroblast differentiation, overall cell density and cell proliferation to be not different between groups, indicating a normal wound healing response after MI [29]. These results suggest that FAP is not crucial for cell proliferation, adherence and migration within the myocardium after MI.
Of note, FAP is expressed in atherosclerotic plaques but its role in regulating inflammatory and fibrotic response is still poorly understood [7,8,31]. Two recent studies reported contrasting data: Monslow et al. demonstrated that global deletion of FAP in ApoE −/− mice accelerated atherosclerotic disease progression by altering macrophage infiltration into the vulnerable plaque [31]. Instead, Stein et al. reported that deletion of FAP in ApoE −/− mice resulted in decreased atherosclerotic plaque formation [8]. These diverging results show that while FAP has a role in atherosclerotic plaque progression, the mechanisms involved are not yet fully understood. Together with our findings we assume with reason that other factors, including well-known matrix degrading enzymes such as matrix-metalloproteinases are involved together with FAP in regulating myocardial ECM content and cell migration in the heart after MI, compensating the loss of FAP in the heart in FAP deficient mice [32].

Study limitations
A main limitation of the study was the relatively low number of mice and few time points to be analyzed. Moreover, we only studied mice at 7 and 28 days after MI. Collagen accumulation extends with time [33], and there might be a difference in ventricular collagen content at later timepoints between WT and FAP-KO animals. Likewise, we did not detect differences in heart failure symptoms, ventricular rupture and mortality between groups. A longer study period extending 28 days post-MI might have shown a beneficial effect of genetic FAP deletion on symptoms of heart failure, ventricular rupture or mortality. In this study we only compared infarcted wild type and FAP-KO mice, but a sham group without MI is missing.
Our results do not reveal a mechanistic cause for the improved remodeling in FAP-KO mice, and future studies are needed to assess the clinical value, if any, of FAP deletion after MI. Because collagen metabolism and ventricular remodeling are different between species [34], studies of FAP depletion in larger animal models are necessary.

Conclusion
The aim of the present study was to analyze whether FAP deficiency may have a protective effect after MI. In fact, MI in FAP-KO mice was associated with reduced LV dilation and did not negatively impact wound healing.
High left ventricular FAP signal intensities as measured by positron-emission-tomography are associated with cardiovascular and metabolic risk factors such as hypertension, diabetes mellitus and obesity [35]. Moreover, advances have been made using therapies depleting FAP cells to treat cancer disease, which could potentially affect the heart [16,36]. Because therapies targeting FAP pos (myo)fibroblasts will likely alter myocardial FAP levels, it is of importance to understand the function and pathophysiological significance of FAP deficiency in normal healthy hearts and post-MI in vivo.
Here, we describe for the first time that genetic ablation of FAP has beneficial effects after MI, and does not alter myocardial structure in healthy animals until 6 months of age. Our study indicates that new therapies associated with a reduction of myocardial FAP levels are safe and can be further developed. More studies are warranted to evaluate the effect of depleting FAP pos cell populations on cardiac function and healing after MI in larger animal models.
Supporting information S1 Fig. Anatomical and morphometric measurements, collagen content in healthy FAP-KO and WT mice at age of 6 months. Body weight was slightly, but significant less in FAP-KO animals compared to WT (A). However, heart weight corrected for tibia length, as well as LV area and LV cavity area (LV area/LV cavity) were not different between groups (B). Collagen content, as measured by hydroxyproline assay, showed no significant difference between groups (C).