Immunofluorescent staining of CD45 (green) and SMA (red) indicate dual staining (yellow) within areas of infiltration in AngII(D), AngII+mIgG(E)treated myocardium, with representative images shown at 63x and an additional 4x digital magnification of an area of dual staining shown(F). data suggest that myocardial fibrosis was increased by the addition of exogenous non-specific antibodies in an Fc-mediated manner. These findings could have substantial impact on the future experimental design of antibody-based therapeutics. Keywords:Myocardial fibrosis, Collagen, Antibody, Inflammation, AngiotensinII == Background == Despite advances in medical therapy, cardiovascular disease remains the cause of 33% of all deaths in the United States and 28% in Canada [1]. Furthermore, the 5-12 months mortality rate for patients presenting with heart failure is usually 50% [2]. The pathological characteristics and the final pathway leading to heart failure is the development of myocardial fibrosis [3]. Myocardial fibrosis is usually defined as extra deposition of extracellular matrix (ECM) proteins within the myocardium and is a significant contributor to myocardial dysfunction. A common animal model of myocardial fibrosis is usually induction by exposure to AngiotensinII (AngII), Igf1r a vasoregulatory hormone, which results in hypertension-mediated myocardial fibrosis [4,5]. We, and others, have previously reported that myocardial fibrosis after exposure to AngII is usually characterized as early as 3 days by myocardial infiltration of mononuclear cells that have been defined as bone marrow-derived, monocyte lineage, fibroblast progenitor cells (fibrocytes) [6-8]. This is followed by a significant increase in extracellular matrix deposition, the hallmark of fibrosis. Using this model, investigators have identified the pro-fibrotic mediator transforming growth factor- (TGF) as being central to the regulation of fibrosis development. In the AngII-induced model, the TGF pathway, including SMAD phosphorylation and CTGF production, are significantly upregulated within the first 3 days, suggesting their involvement in the initial phase of myocardial fibrosis development [4]. In support of the role for TGF, inhibition of TGF using antibodies [9] or mice heterozygous for TGF knockout [10], have resulted in reduced myocardial fibrosis. However, TGF Tiplaxtinin (PAI-039) is a pleiotropic cytokine that has both pro-inflammatory and anti-inflammatory functions, making treatment strategies based on its reduction difficult, as serious deleterious effects have been reported, such as increased mortality after myocardial infarction [11]. Other antibody-based therapeutic strategies attempted in animal models have included targeting IL23R, platelet-derived growth factor-receptor, and the TGF downstream mediator connective tissue growth factor (CTGF), without any antibody-based therapy in clinical use to date [12,13]. In the present study, we show that non-specific antibody, similar to those used as controls for therapies used to treat myocardial fibrosis can Tiplaxtinin (PAI-039) in fact worsen the onset of fibrosis in the AngII-induced model. We provide evidence that this mechanism for this apparent paradoxical observation is usually mediated by increased pro-inflammatory cytokine production and is not dependent on the specificity of the antibody. In fact, the worsened fibrotic response to whole antibody Tiplaxtinin (PAI-039) disappeared with treatment using Fab fragments, suggesting that an Fc-mediated activation of infiltrated cells was key to the exacerbated fibrotic response. == Results == == Effect of antibody on early myocardial fibrosis == The addition of non-specific antibody by tail-vein injection to the AngII model of myocardial fibrosis over 3 days resulted in a significant increase of the fibrotic area within the myocardium. The amount of fibrosis was significantly increased after mIgG treatment, with 26.34 3.03% of myocardium affected compared to 11.29 1.20% with AngII alone, and with a more dispersed pattern of collagen deposition (Determine1A, C, D). The mRNA expression ofCol1A1, a subunit of type I collagen, the most prolific structural collagen in the myocardium, also increased in the myocardium of animals exposed to AngII, but did not increase further in the.