Role of Pathologic Shear Stress Alterations in Aortic Valve Endothelial Activation
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- Hoehn, D., Sun, L. & Sucosky, P. Cardiovasc Eng Tech (2010) 1: 165. doi:10.1007/s13239-010-0015-5
Calcific aortic stenosis is the most common aortic valve (AV) disease and is triggered by an active inflammatory process involving endothelial activation and cytokine expression. Interfacing between the leaflet and the surrounding blood flow, shear stress is presumed to play an important role in endothelial injury. This study investigated the hypothesis that pathologic alterations in shear stress magnitude contribute to valvular endothelial activation via BMP-4- and TGF-β1-dependent mechanisms. The fibrosa of porcine AV leaflets was subjected to physiologic, sub-physiologic and supra-physiologic magnitudes of native oscillatory shear stress for 48 h. Endothelial activation was assessed via immunohistochemistry in terms of ICAM-1 and VCAM-1 expressions. Cytokine expression was investigated in terms of BMP-4 and TGF-β1. Pro- and anti-osteogenic media were used to characterize the role of those cytokines in the shear stress-induced pathological response. Supra-physiologic shear stress increased the expression of all biomarkers in a shear stress magnitude-dependent manner. In contrast, neither physiologic nor sub-physiologic shear stress elicited a pro-inflammatory response. While BMP-4 inhibition and supplementation had limited effects on endothelial activation, TGF-β1 supplementation increased the overall leaflet pro-inflammatory state and TGF-β1 inhibition reduced endothelial activation in response to elevated shear stress. Combined TGF-β1 and BMP-4 inhibition completely suppressed shear stress-induced endothelial activation. The results demonstrate that elevated shear stress activates the valvular endothelium on the fibrosa via a BMP-4- and TGF-β1-dependent pathway. The suggested synergy between those cytokines also provides new insights into the transduction of valvular hemodynamic alterations into a pathological response.
KeywordsAortic valveEndothelial activationShear stressCytokinesAdhesion molecules
Calcific aortic stenosis is the most prevalent aortic valve (AV) disease and is present in 8% of the population above 65 years of age.44 The side-specific formation of calcific nodules on the aortic surface of the leaflets14,32,34 contributes to the obstruction of the left ventricular outflow and can lead ultimately to heart failure. The previously accepted theory that linked valvular calcification to a passive wear-and-tear mechanism has lost support due to new developments that have associated the disease with an active process involving inflammation and ossification.17,26,31,38,39 The characterization of calcific lesions suggests that the early stage of the disease is marked by cell proliferation and increased expressions of adhesion molecules, bone morphogenic proteins (BMP) and transforming growth factors-beta (TGF-β).20,30,39 Although the inflammatory stage is thought to be associated with the dysfunction of the leaflet endothelium,42,43 the mechanisms contributing to endothelial activation are not well understood.
Clinical observations, animal and ex vivo studies have suggested that the hemodynamic stress environment experienced by the leaflets may regulate valvular physiology and pathology.15,31,40,41,48,51 Resulting from the relative motion between the leaflet surface and the surrounding pulsatile blood flow, shear stress is an important component of the valve hemodynamic environment.40 The particular valve anatomy and leaflet dynamics give rise to a side-specific shear stress defined by a high unidirectional pulsatile shear stress along the ventricular leaflet surface (ventricularis) and a low bidirectional oscillatory shear stress along the aortic surface (fibrosa).24,49 This complexity has hampered our understanding of the biological processes regulated by the native valvular fluid shear stresses. Although studies have demonstrated the characteristic alignment of valvular endothelial cells perpendicular to steady unidirectional flow5 and the dependence of valvular remodeling activity on steady shear stress magnitude,36 studies on the effects of pathologically relevant shear stress alterations on valvular biology are few. A previous study on the effects of the native oscillatory and pulsatile fluid shear stresses on AV inflammation indicated that simultaneous alterations in shear stress magnitude and pulsatility on the fibrosa could trigger the activation of the endothelium and the upregulation of the cytokines BMP-4 and TGF-β1.45 The significant downregulation of this response following the pharmacological inhibition of BMP-4 or TGF-β1 also suggested a role for those mediators in the shear stress-induced inflammatory pathway.
Although those findings demonstrate the existence of relationships between flow alterations and valvular inflammation, their translation to disease initiation and progression is limited since pathologic valvular hemodynamics is unlikely to result in simultaneous alterations in both shear stress pulsatility and magnitude. For example, the accelerated progression of calcific aortic stenosis19 is accompanied by a reduction in valvular effective orifice area which leads, in turn, to an increased peak aortic velocity.9,33 Similarly, hypertension (i.e., a risk factor for calcific aortic stenosis37) is associated with changes in transvalvular flow rate,21,23,28 which also translates in variations in peak aortic velocity. Those observations may indicate alterations in shear stress magnitude as a key mechanism in the development of valvular disease. Therefore, this study hypothesized that pathological alterations in shear stress magnitude promote valvular endothelial activation via BMP-4- and TGF-β1-dependent mechanisms. This hypothesis was tested via four experiments aimed at: characterizing the effects of normal and pathologic shear stress magnitudes on the expressions of cytokines (BMP-4, TGF-β1) and cell adhesion molecules (ICAM-1, VCAM-1) associated with valvular endothelial activation (experiment 1); investigating the respective role played by BMP-4 and TGF-β1 in valvular endothelial activation in response to pathologic shear stress levels (experiments 2 and 3, respectively); and exploring the synergy between those two cytokines in response to pathologic shear stress magnitudes (experiment 4).
Materials and Methods
Experimental Groups and Conditions
Experiments and treatment groups considered in this study
DMEM + noggin
DMEM + BMP-4
DMEM + SB-431542
DMEM + TGF-β1
DMEM + noggin + SB-431542
Porcine hearts were obtained from a local slaughterhouse (Martin’s Custom Butchering, Wakarusa, IN), immediately rinsed in sterile Dubelcco’s phosphate buffered saline (PBS, Sigma-Aldrich) and transported to the laboratory in ice-cold PBS. All subsequent procedures were carried out in a sterile flow hood. Each group consisted of nine circular leaflet samples excised from the basal leaflet region. The nine samples assigned to each treatment group were randomized and selected from different animals. Fresh leaflet samples from the control group (group 1) were processed immediately as outlined in “Immunohistochemical Analysis” section, while samples from the six other groups were conditioned to shear stress in a cone-and-plate bioreactor described and validated previously.46 The whole setup was placed in an incubator at 37 °C and 5% CO2. Tissue from groups 2 to 7 was exposed to shear stress for 48 h, a duration sufficient for biological changes in response to mechanical stimulation to become evident.1,2,27,45,52 Culture medium was continuously perfused during each experiment at a rate of 82 mL/h (i.e., one bioreactor volume/hour) and visually inspected for evidence of contamination.
The objectives of the biological analyses were 2-fold: (1) to assess whether specific markers associated with endothelial activation were expressed in response to pathologic shear stress levels; and (2) to determine their specific site of expression in the tissue. Immunohistochemistry, which is able to provide such information in one single step, was performed for all analyses. Following shear stress conditioning, tissue samples from groups 2 to 7 were harvested and washed immediately in sterile PBS. For each specimen, the region exposed to flow was separated from the peripheral region that was clamped to maintain the sample in position during the experiments. The specimens were embedded in optimal cutting temperature compound and flash frozen in liquid nitrogen. Tissue sections were then mounted on slides and stored in a −80 °C freezer. Standard immunostaining procedures were used to identify cells positive for VCAM-1 (1:50, Santa Cruz), ICAM-1 (1:50, SouthernBiotech), TGF-β1 (1:25, Santa Cruz), and BMP-4 (1:25, Santa Cruz). Tissue was also probed with vWF (1:200, Sigma) to identify endothelial phenotype and to assess endothelium integrity. Detection of cell apoptosis was performed by a TUNEL assay (Roche Diagnostics).
The intensities of BMP-4-, ICAM-1-, VCAM-1-, and TGF-β1-positive green stains were estimated using Image J (NIH, Bethesda, MD) and normalized by the number of cells visible in each microscope image to yield a quantity consistent to an expression per cell. The number of cells was estimated by counting the number of DAPI-positive nuclei imaged by ultra-violet (UV) epifluorescence. The total expression of a given molecule was assessed by quantifying the intensity of the FITC-positive stain on each image, which was done by computing the integral of the green channel histogram. Cellular expression was calculated as the ratio of the total expression of a given molecule to the number of cells in each image. Apoptosis level was estimated by the ratio of the number of cells with apoptotic fragments as detected under UV epifluorescence using the FITC filter to the total number of cells present in each image.
All quantitative data were expressed as mean ± standard error. The sample size for each experimental group was n = 9. For each experimental condition, the semi-quantitative results were averaged over the nine samples to provide a mean cellular expression. All results were then normalized with respect to the values measured in fresh tissue (group 1) to yield a fold increase with respect to fresh tissue. The data were first analyzed using ANOVA to determine if there was significant contribution by a particular treatment on the measured parameters, followed by the Tukey post hoc test. A p-value of less than 0.05 was used as a measure of statistical significance. All statistical analyses were performed using Minitab 16 (Minitab Inc).
Sub- and Supra-Physiologic Shear Stresses Maintain Endothelium Integrity and Cell Viability
Elevated Shear Stress Increases Expression of Pro-Inflammatory Markers on the Fibrosa in a Magnitude-Dependent Manner
BMP-4 Regulates Adhesion Molecule Expression on the Fibrosa Exposed to Pathologic Shear Stress
In order to determine the role played by BMP-4 in the shear stress-induced valvular endothelial activation (experiment 2), the fibrosa was exposed to elevated (mild and severe supra-physiologic) shear stress in culture medium supplemented with either the BMP antagonist noggin (group 3) or BMP-4 (group 4).
BMP Inhibition Reduces TGF-β1 and Adhesion Molecule Expressions in Response to Elevated Shear Stress
BMP-4 Supplementation Does Not Affect Shear Stress-Induced Endothelial Activation
TGF-β1 Regulates Adhesion Molecule and BMP-4 Expressions on the Fibrosa Exposed to Pathologic Shear Stress
The role played by TGF-β1 in the shear stress-induced valvular endothelial activation (experiment 3) was investigated by supplementing the standard culture medium with either the specific TGF-β1 inhibitor SB-431542 (group 5) or TGF-β1 (group 6). The fibrosa was exposed to mild and severe supra-physiologic shear stress magnitudes for 48 h.
TGF-β1 Inhibition Prevents Shear Stress-Induced Endothelial Activation
TGF-β1 Supplementation Increases Endothelial Activation in Response to Severe Supra-Physiologic Shear Stress
Combined BMP-4 and TGF-β1 Inhibition Suppresses Cell Adhesion Molecule and Cytokine Expressions on the Fibrosa Exposed to Elevated Shear Stress
The objectives of this study were to explore the acute effects of sub- and supra-physiologic shear stress on valvular endothelium activation, and to characterize the role played by the cytokines BMP-4 and TGF-β1 in that response. The contributions can be summarized under the following three points: (1) exposure of the fibrosa to supra-physiologic levels of shear stress stimulates the expressions of cytokines and cell adhesion molecules within 48 h; (2) the cytokines BMP-4 and TGF-β1 interact to synergistically regulate endothelial activation in response to elevated shear stress; and (3) TGF-β1 has more effect on the shear stress-induced pathological response than BMP-4.
Role of Endothelium in Valvular Pathogenesis
The absence of cytokine and adhesion molecule expression following the exposure of the fibrosa to its physiologic level of bidirectional oscillatory shear stress is consistent with previous studies that demonstrated the key role of physiologic hemodynamic stresses in preserving valvular function.1,50 The onset of a pathological response localized in the endothelial and sub-endothelial layers after exposure of the fibrosa to elevated shear stress magnitudes is also supported by previous results that demonstrated the ability of drastic alterations in shear stress pulsatility to induce a pro-inflammatory response.45 Therefore, this study provides new evidence that the fibrosa is sensitive to its hemodynamic environment and is able to transduce alterations in both shear stress pulsatility and magnitude into a pro-inflammatory response. The detection of cytokine and adhesion molecule expression in the endothelial layer of the fibrosa after exposure to elevated shear stress is consistent with the known athero-prone transcriptional profile of this endothelium43 and indicates the critical role played by the valve endothelial cell population in the transduction of abnormal hemodynamic stimuli into a pathological state.
The results of this study provide further evidence of the distinct differences between the valvular and vascular endothelia.6,11 While regions of the vasculature exposed to low oscillatory shear stress are prone to atherosclerosis and plaque formation,7,8 the present results demonstrate that only supra-physiologic levels of oscillatory shear stress trigger the acute activation of the endothelium lining the aortic surface of the valve leaflets. On the other hand, neither physiologic nor sub-physiologic oscillatory shear stress level elicited a similar pathological response. Therefore, the present findings tend to lend less credibility to the atherosclerotic root of valvular calcification and suggest the involvement of environmental cues such as hemodynamic alterations in valvular pathogenesis.
Shear Stress in Valvular Disease
Although the shear stress alterations considered in this study have not been measured under native disease conditions, the results may yet be pathophysiologically relevant. In fact, the progression of calcific aortic stenosis is accompanied by an increased peak aortic velocity,33,35 which could translate into a 2-fold and 4-fold increase in valvular wall-shear stress magnitude under mild and severe stenotic conditions, respectively. Therefore, the results, which suggest an increased pathological state under elevated shear stress, could also explain the accelerated progression of valvular calcification in patients with stenotic19 and bicuspid4 AVs. Studies on the characterization of the hemodynamic forces experienced by AV leaflets under diseased conditions are currently under way in our laboratory in order to study the impact of pathologically relevant shear stress conditions on valvular inflammation and calcification. Specifically, we are developing computational and experimental methodologies based on fluid structure interaction modeling and particle-image velocimetry, respectively, to capture the native valvular stress state under normal and disease conditions (e.g., bicuspid aortic valve, calcified aortic valve). This hemodynamic characterization will permit to investigate the effects of the native disease-induced shear stresses on valvular pathobiology using the same ex vivo approach as that described in this study.
It is important to note that, although valvular calcific degeneration occurs over a much longer timescale than that considered in this study, the present results demonstrate the existence of acute biological changes in response to mechanical stimulation. Those acute effects are important as they may shape the longer-term mechanisms involved in the progression of valvular disease. Therefore, a complete understanding of valvular degenerative processes can only be obtained by considering both the acute and long-term response to mechanical cues. A new shear stress bioreactor capable of maintaining fresh leaflet tissue under a near-native biomechanical environment for longer periods is being designed in our laboratory and will provide more insights into valvular pathogenesis.
Potential Mechanosensitivity of TGF-β1 and BMP-4
An important contribution of this study is the confirmation of the global decrease in shear stress-induced valvular endothelial activation after silencing TGF-β1 and BMP-4. While the BMP antagonist noggin and the TGF-β1 inhibitor SB-431542 each resulted in a decrease in fibrosa activation following exposure to elevated shear stress, the combined inhibition of those cytokines completely suppressed the shear stress-induced pathological response and returned adhesion molecule expressions to levels similar to those detected in fresh tissue. This observation suggests the key role played by those cytokines and their synergy in the transduction of pathologic hemodynamic alterations into a pro-inflammatory response. This notion is supported by numerous studies that have identified TGF-β1 and BMPs as molecules involved in the early stage of valvular calcification,16,22,29,47 and studies that have demonstrated their side-specific expression on the disease-prone fibrosa.43 Importantly, the detection of TGF-β1 and BMP-4 expression in both the endothelial and subendothelial layers of the fibrosa and the clear dependence of the shear stress-induced endothelial activation on those molecules suggest the mechanosensitivity of those cytokines and their ability to transduce abnormal hemodynamic stresses into a pro-inflammatory response on the fibrosa. Although more studies are needed to confirm this hypothesis, the present results clearly demonstrate strong interactions between BMP-4 and TGF-β1 in valvular pathogenesis.
Model of BMP-4/TGF-β1 Synergy in Shear-Induced Valvular Endothelial Activation
This study provides new evidence of the key role played by abnormal hemodynamic forces in the progression of valvular disease. The results suggest the involvement of the leaflet endothelium and the cytokines TGF-β1 and BMP-4 in the transduction of fluid shear stress alterations into a pro-inflammatory response. A more detailed characterization of the molecular mechanisms mediated by TGF-β1 and BMP-4 is needed for the development of targeted pharmacological modalities aimed at preventing the onset or slowing the progression of valvular calcification.
The authors thank Steven DeLaurentis and Michael O’Connor (University of Notre Dame) for their assistance with the experiments, Leon Hluchota (University of Notre Dame) for his advice on the bioreactor design and fabrication, and Martin’s Custom Butchering (Wakarusa, IN) for supplying porcine hearts. This work was supported by the Faculty Research Program at the University of Notre Dame.