Faculty Bibliography

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis.

Chronic rejection significantly limits long-term success of solid organ transplantation. De novo donor-specific antibodies (DSAs) to mismatched donor human leukocyte antigen after human lung transplantation predispose lung grafts to chronic rejection. We sought to delineate mediators and mechanisms of DSA pathogenesis and to define early inflammatory events that trigger chronic rejection in lung transplant recipients and obliterative airway disease, a correlate of human chronic rejection, in mouse. Induction of transcription factor zinc finger and BTB domain containing protein 7a (Zbtb7a) was an early response critical in the DSA-induced chronic rejection. A cohort of human lung transplant recipients who developed DSA and chronic rejection demonstrated greater Zbtb7a expression long before clinical diagnosis of chronic rejection compared to nonrejecting lung transplant recipients with stable pulmonary function. Expression of DSA-induced Zbtb7a was restricted to alveolar macrophages (AMs), and selective disruption of Zbtb7a in AMs resulted in less bronchiolar occlusion, low immune responses to lung-restricted self-antigens, and high protection from chronic rejection in mice. Additionally, in an allogeneic cell transfer protocol, antigen presentation by AMs was Zbtb7a-dependent where AMs deficient in Zbtb7a failed to induce antibody and T cell responses. Collectively, we demonstrate that AMs play an essential role in antibody-induced pathogenesis of chronic rejection by regulating early inflammation and lung-restricted humoral and cellular autoimmunity.

Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFkappaB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFkappaB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IkappaBalpha, a member of the protein complex inhibiting NFkappaB activation. Binding of IkappaBalpha (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFalpha-induced nuclear translocation of NFkappaB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFalpha-induced NFkappaB phosphorylation. Finally, Tie2CreTgCx40fl/flApoe-/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFkappaB nuclear translocation. Our data show a novel functional IkappaBalpha-Cx40 interaction that may be relevant for the control of NFkappaB activation by shear stress in atherogenesis.

Cancers are infiltrated with antitumor CD8+ T cells that arise during tumor growth, but are defective in effector phase functions because of the suppressive microenvironment. The reactivation of TILs can result in tumor destruction, showing that lytic dysfunction in CD8+ tumor-infiltrating lymphocytes (TILs) permits tumor growth. Like all memory T cells, TILs express inhibitory signaling receptors (aka checkpoint inhibitor molecules) that downregulate TCR-mediated signal transduction upon TIL interaction with cells expressing cognate ligands, thereby restricting cell activation and preventing the effector phase. Previously we identified a novel murine CD8+ TIL inhibitory signaling receptor, protocadherin-18, and showed that it interacts with p56lck kinase to abrogate proximal TCR signaling. Here we show that TILs from mice deleted in protocadherin-18 had enhanced antitumor activity and that co-blockade of PD-1 and protocadherin-18 in wild-type mice significantly enhanced TIL effector phase function. These results define an important role for protocadherin-18 in antitumor T-cell activity.

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Background: Arrhythmogenic cardiomyopathy (also known as "ARVC") is an inherited disease characterized by fibrous or fibrofatty infiltration of the heart muscle, commonly of right ventricular (RV) predominance, ventricular arrhythmias, and high propensity for sudden death. Sudden cardiac arrest frequently associates with exercise and most often occurs in early adulthood during the subclinical ("concealed") phase of the disease. Understanding electrical remodeling in the early stage of the disease is paramount to understand sudden death mechanisms. Methods: We generated a cardiomyocyte-specif-ic, tamoxifen-activated, PKP2 knockout murine line (alphaMHC-Cre-ERT2/PKP2 fl/fl) which allowed us to control the onset of PKP2 loss of expression, limit it to adult cardiomyocytes, and establish a time line for progression of molecular and functional events. Results: The first consequence of PKP2 loss was RV mechanical dysfunction (14 days post-tamoxifen injection, 14 dpi), followed by fibrosis of RV predominance and RV dilation (21 dpi), then biventricular dilated cardiomyopa-thy and left ventricular (LV) failure (28 dpi and beyond). End-stage failure and death occurred between 30 and 49 dpi. Isoproterenol (ISO)-induced ventricular arrhythmias were first detected prior to LV dysfunction (17/17 mice), and ISO-induced fatal ventricular fibrillation was observed only at 16 dpi, i.e., during the concealed stage (3/9). Differential tran-scriptome analysis at 21 dpi revealed reduced transcript levels for a gene network involved in intracellular calcium ([Ca²⁺]i) cycling, most critically genes encoding Ca²⁺ channel proteins (RyR2 and CaV1.2) and structural molecules that scaffold the dyad (ankyrin-B and triadin). Nanoscale imaging (3D super-resolution microscopy, SICM, and FIB-SEM) showed preservation of T-tubular structure, reduced size and increased separation of CaV1.2 clusters, and displacement of functional CaV1.2 channels from the T-tubular domain. Calcium imaging showed disruption of [Ca²⁺]i homeostasis, potentially causative of ventricular arrhythmias. Flecainide i.p. prevented ISO-induced arrhythmias in all animals. Retrospective analysis of clinical cases showed instances of sudden cardiac arrest without structural disease and suspect diagnosis of catechol-aminergic polymorphic ventricular tachycardia (CPVT) later revealed to foster PKP2 nonsense mutations. Conclusions: Our data provide the first evidence that PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmo-genic cardiomyopathy of RV predominance. Adrenergic-induced arrhythmias and sudden death occur before the onset of overt structural disease and can mimic a CPVT phenotype. Our data also document a transcript-based [Ca²⁺]i dysfunction as a new key mechanism of arrhythmias in PKP2-deficient hearts and suggest flecainide as potential effective antiarrhyth-mic treatment