Fibrotic disease affects both the heart valves and the heart myocardium and is characterized by changes in the mechanical properties of the ECM and the cellular phenotypes present in the tissue. In both chronic conditions and MI, inflammatory cytokines promote tissue remodeling and ECM degradation in the myocardium [71] , [72] . However, excessive TGF-β1 signaling has been implicated in another commonly observed example of maladaptive remodeling.
The convergence on TGF-β1 signaling is just one example of the significant crosstalk between integrin and growth factor signaling involved in the regulation of MyoFB differentiation in the heart (Figure 3.2) [92]. Cadherin-2 (N-cadherin) is the classical cell-cell adhesion protein expressed by quiescent fibroblasts and CMs in the heart. Cadherin-11 (also known as OB-cadherin) was first described in 1994 in the context of osteogenesis and bone development, but recent work has shown its importance in a range of tissues [161].
In the context of cancer, cadherin-11 expression is associated with increased migration and metastasis, particularly metastasis to bone [162]. In this context, cadherin-11 has been implicated in the progression of dermal and pulmonary fibrosis and suggested to promote MyoFB differentiation through interactions with β-catenin. Taken together, these studies suggest that cadherin-11 is a promising target in the field of fibrotic disease.
During MyoFB differentiation, TGF-β1 signaling induces an increase in cadherin-11, concomitant with a decrease in cadherin-2 expression [44].
ELUCIDATING MECHANOSENSITIVE SIGNALING CROSSTALK IN FIBROBLASTS
The objectives of this goal are to clarify the roles of FAK and Src in coupling integrin and cytokine signaling, and to characterize the signaling profiles of TGF-β1 and FGF2 through p38 and ERK in the regulation of α-SMA and cadherin 11 expression. Cadherin-11 expression was significantly lower in FAK -/- cells at all stiffnesses and is less sensitive to changes in stiffness than α-SMA (Figure 4.6B). We observed a significant decrease in cadherin-11 expression from TCP to 10:1 PDMS in MEF+/+ cells.
The apparent importance of FAK in both α-SMA and cadherin-11 regulation suggests an important regulatory role for ERK. In MEFs, we observed a significant increase in both cadherin-11 expression and α-SMA with LiCl alone. We also observed a decrease in cadherin-11 expression with at 900 kPa PDMS relative to plastic, although there was no significant change in FAK-/- cells.
Interestingly, in both MEF+/+ and FAK-/- cells, cadherin-11 expression appears to trend upwards on softer PDMS samples. GSK-3β has been reported to regulate cadherin-11 expression [205] and is known to play important roles in healing.
CADHERIN-11 EXACERBATES TISSUE REMODELING AFTER MYOCARDIAL INFARCTION
Established inflammation-induced fibrosis in the lungs has been reversed by treatment with functional blocking antibody against cadherin-11 [25]. Cadherin-11 (CDH11) transcription was significantly higher in the non-cardiomyocyte (non-CM) cell population compared to cardiomyocytes (CM) (* indicates p < 0.05) (A). Immunostaining confirmed the presence of cadherin-11-expressing Ms, endothelial cells and MyoFBs in the infarct region seven days after MI (D).
These results confirm the functional role of cadherin-11 in the process of myocardial remodeling after MI. Immunostaining of MyoFB markers in the infarct at twenty-one days (C) and trichrome images of the infarct (yellow dashed area indicates the approximate area of the images in C) to show scar thickness (D). Western blot showed that α-SMA was significantly decreased twenty-one days after infarction in treated animals (E).
We observed a decrease in transcription of IL-6, a pro-inflammatory cytokine, in the antibody-treated group three days after MI (Figure 5.6A). This reduction appears to occur primarily in the non-CM cells of the infarct, a mixed population of inflammatory cells, endothelial cells and myofibroblasts (Figure 5.6B). Immunostaining of hearts on day three post-infarction shows an apparent reduction in the colocalization of (and thus the likely interactions between) activated MyoFBs (α-SMA) and Ms (F4/80) in the infarct and border zone (Figure 5.7B ) ).
Regarding profibrotic markers, note that TGF-1 transcription is no longer significantly increased in the SYN0012-treated group seven days after MI in contrast to the control group (Figure 5.9A). This observed decrease in MyoFB in the infarct correlates well with the decrease in α-SMA and reduced scar compaction (Figure 5.4B-E). 1, transcription was significantly enhanced by co-culture and, somewhat surprisingly, also significantly enhanced by SYN0012 in the co-culture setting (Figure 5.12A).
Immunostaining revealed that a subset of Ms, endothelial cells, smooth muscle cells, and MyoFB in the infarcted region at seven days post-MI all express cadherin-11. The results indicated that cadherin-11 may play a functional role in increasing LV remodeling after MI during the first seven days. Using AFM, we observed changes in the mechanical environment of the infarct during infarct healing.
To this end, we performed co-culture experiments to determine whether cadherin-11 can mediate interactions between M and MyoFB in the regulation of inflammatory and fibrotic signaling. This study led us to develop the following possible cellular mechanism for the role of cadherin-11 in infarct remodeling (Figure 5.14).
QUANTIFYING CARDIOMYOCYTE MECHANICS
The lateral deflection of the probe tip (𝛥𝑠 ) and the lateral displacement of the center of the construct (𝛥𝑠 ) add up to the total deflection of the table (𝛥𝑠. Passive model parameters were estimated for each construct using passive force linear least squares analysis. Furthermore, averaging the estimated parameters across constructs gave a robust fit average developed forces (Figure 6.7F).
The experimental results revealed an overall decrease in the maximum force developed, as well as a flattening of the downward trend in force generation (Figure 6.9B). Mechanobiology is particularly relevant in the heart because of the unique mechanical demands of cardiac function and the dynamic process of tissue remodeling (and subsequent alteration of mechanical signals) that occurs during disease. We observed a significant decrease in the functional viscosity of the constructs in response to isoproterenol treatment.
My findings regarding regulation of the MyoFB phenotype will inform future studies of integrin signaling and. Two of the most likely candidates for signaling downstream of cadherin-11 are β-catenin and p120 catenin. The phosphorylated form of the receptor (pTBR2) is able to induce p38 phosphorylation and Src activation [177] (Equation A6.4).
These data indicate that the features of the model presented above have reasonable support from the data. Sensitivity analysis of the model predicted higher sensitivity to FGF2 and stiffness in FAK-/- cells compared to MEF+/+. FC force of I-Wire construct in response to FP (N) preload Estimated stress in unstretched construct (N).
The force in the I-wire construct is the sum of the forces in the passive non-actin/myosin elements ( ), associated with the extracellular matrix, and in the myocytes themselves. The same force passes through the left (parallel) and right (serial) components of the myocyte's contribution. Shai et al., "Cardiac myocyte-specific excision of the ??1 integrin gene results in myocardial fibrosis and cardiac failure," Cir.
Penna et al., "Overexpression of the muscle-specific protein melusin protects against cardiac ischemia/reperfusion injury," Basic Res Cardiol, vol. Hanafusa et al., "Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta- induced gene expression,” J Biol Chem , vol.
