Hayden Huang

The Impact of Calcification on the Biomechanical Stability of Atherosclerotic Plaques

Background —Increased biomechanical stresses in the fibrous cap of atherosclerotic plaques contribute to plaque rupture and, consequently, to thrombosis and myocardial infarction. Thin fibrous caps and large lipid pools are important determinants of increased plaque stresses. Although coronary calcification is associated with worse cardiovascular prognosis, the relationship between atheroma calcification and stresses is incompletely described. Methods and Results —To test the hypothesis that calcification impacts biomechanical stresses in human atherosclerotic lesions, we studied 20 human coronary lesions with techniques that have previously been shown to predict plaque rupture locations accurately. Ten ruptured and 10 stable lesions derived from post mortem coronary arteries were studied using large-strain finite element analysis. Maximum stress was not correlated with percentage of calcification, but it was positively correlated with the percentage of lipid (P =0.024). When calcification was eliminated and replaced with fibrous plaque, stress changed insignificantly; the median increase in stress for all specimens was 0.1% (range, 0% to 8%;P =0.85). In contrast, stress decreased by a median of 26% (range, 1% to 78%;P =0.02) when lipid was replaced with fibrous plaque. Conclusions —Calcification does not increase fibrous cap stress in typical ruptured or stable human coronary atherosclerotic lesions. In contrast to lipid pools, which dramatically increase stresses, calcification does not seem to decrease the mechanical stability of the coronary atheroma.

A Three-Dimensional Viscoelastic Model for Cell Deformation with Experimental Verification

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending <10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is >5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.

Transcriptional Profile of Mechanically Induced Genes in Human Vascular Smooth Muscle Cells

Vascular smooth muscle cells must monitor and respond to their mechanical environment; however, the molecular response of these cells to mechanical stimuli remains incompletely defined. By applying a highly uniform biaxial cyclic strain to cultured cells, we used DNA microarray technology to describe the transcriptional profile of mechanically induced genes in human aortic smooth muscle cells. We first identified vascular endothelial growth factor (VEGF) as a mechanically induced gene in these cells; VEGF served as a positive control for these experiments. We then used a DNA microarray with 5000 genes with putative functions to identify additional mechanically induced genes. Surprisingly, relatively few genes are mechanically induced in human aortic smooth muscle cells. Only 3 transcripts of 5000 were induced >2.5-fold: cyclooxygenase-1, tenascin-C, and plasminogen activator inhibitor-1. Downregulated transcripts included matrix metalloproteinase-1 and thrombomodulin. The transcriptional profile of mechanically induced genes in human aortic smooth muscle cells suggests a response of defense against excessive deformation. These data also demonstrate that in addition to identifying large clusters of genes that respond to a given stimulus, DNA microarray technology may be used to identify a small subset of genes that comprise a highly specific molecular response.

Thioredoxin-Interacting Protein Controls Cardiac Hypertrophy Through Regulation of Thioredoxin Activity

Background—Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. Methods and Results—Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127±5% of controls, P < 0.01). Overexpression of thioredoxin-interacting protein (Txnip), an endogenous thioredoxin inhibitor, reduced protein synthesis in response to mechanical strain (89±5% reduction, P < 0.01), phenylephrine (80±3% reduction, P < 0.01), or angiotensin II (80±4% reduction, P < 0.01). In vivo, myocardial thioredoxin activity increased 3.5-fold compared with sham controls after transverse aortic constriction (P < 0.01). Aortic constriction did not change thioredoxin expression but reduced Txnip expression by 40% (P < 0.05). Gene transfer studies showed that cells that overexpress Txnip develop less hypertrophy after aortic constriction than control cells in the same animals (28.1±5.2% reduction versus noninfected cells, P < 0.01). Conclusions—Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening.

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

A dynamic zone defines interneuron remodeling in the adult neocortex

The contribution of structural remodeling to long-term adult brain plasticity is unclear. Here, we investigate features of GABAergic interneuron dendrite dynamics and extract clues regarding its potential role in cortical function and circuit plasticity. We show that remodeling interneurons are contained within a “dynamic zone” corresponding to a superficial strip of layers 2/3, and remodeling dendrites respect the lower border of this zone. Remodeling occurs primarily at the periphery of dendritic fields with addition and retraction of new branch tips. We further show that dendrite remodeling is not intrinsic to a specific interneuron class. These data suggest that interneuron remodeling is not a feature predetermined by genetic lineage, but rather, it is imposed by cortical laminar circuitry. Our findings are consistent with dynamic GABAergic modulation of feedforward and recurrent connections in response to top-down feedback and suggest a structural component to functional plasticity of supragranular neocortical laminae.

Regulation of Cardiomyocyte Mechanotransduction by the Cardiac Cycle

Background —Overloading the left ventricle in systole (pressure overload) is associated with a distinct morphological response compared with overload in diastole (volume overload). Methods and Results —We designed a novel computer-controlled experimental system that interfaces biaxially uniform strain with electrical pacing, so that cellular deformation can be imposed during a specified phase of the cardiac cycle. Cardiomyocytes were exposed to strain (4%) during either the first third (systolic phase) or last third (diastolic phase) of the cardiac cycle. Strain imposed during the systolic phase selectively activated p44/42 mitogen-activated protein kinase (MAPK) and MAPK/extracellular signal–regulated protein kinase kinase (MEK1/2, an activator of p44/42 MAPK) compared with strain imposed during the diastolic phase. In contrast, there was no difference in activation of p38 and c-Jun NH2-terminal kinases induced by strain imposed during the systolic phase (5.8- and 3.3-fold versus control, n=4) compared with the diastolic phase (5.5- and 3.1-fold). Induction of both brain natriuretic peptide (5.8-fold versus control, P <0.05, n=3) and tenascin-C (7.0-fold, P <0.02) mRNA expression by strain imposed during the systolic phase was greater than during the diastolic phase (3.9- and 3.6-fold, respectively). [3H]leucine incorporation induced by strain imposed during the systolic phase (4.0-fold versus control) was greater than during the diastolic phase (2.7-fold, P <0.02, n=4); a selective inhibitor of MEK1/2 inhibited this difference. Conclusions —Mechanical activation of p44/42 MAPK and MEK1/2, gene expression, and protein synthesis is regulated by the cardiac cycle, suggesting that mechanotransduction at the cellular level may underlie differences between pressure and volume overload of the heart.

High-resolution whole organ imaging using two-photon tissue cytometry

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimers disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.

Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

Genetically engineered resistance for MMP collagenases promotes abdominal aortic aneurysm formation in mice infused with angiotensin II.

Clinical evidence links increased aortic collagen content and stiffness to abdominal aortic aneurysm (AAA) formation. However, the possibility that excess collagen contributes to AAA formation remains untested. We investigated the hypothesis that augmented collagen promotes AAA formation, and employed apoE-null mice expressing collagenase-resistant mutant collagen (ColR/R/apoE−/−), heterozygote (ColR/+/apoE−/−), or wild-type collagen (Col+/+/apoE−/−) infused with angiotensin II to induce AAA. As expected, the aortas of ColR/R/apoE−/− mice contained more interstitial collagen than those from the other groups. Angiotensin II treatment elicited more AAA formation in ColR/R/apoE−/− mice than ColR/+/apoE−/− or Col+/+/apoE−/− mice. Aortic circumferences correlated positively with collagen content, determined by picrosirius red and Masson trichrome staining. Mechanical testing of aortas of ColR/R/apoE−/− mice showed increased stiffness and susceptibility to mechanical failure compared to those of Col+/+/apoE−/− mice. Optical analysis further indicated altered collagen fiber orientation in the adventitia of ColR/R/apoE−/− mice. These results demonstrate that collagen content regulates aortic biomechanical properties and influences AAA formation.