Selective αv integrin depletion identifies a core, targetable molecular pathway that regulates fibrosis across solid organs

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases

Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression

Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R0-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor

Multiple Phenotypes in Adult Mice following Inactivation of the Coxsackievirus and Adenovirus Receptor (Car) Gene

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell–cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell–cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell–cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell–cell contact formation and maintenance

Targeting of alpha(v) integrin identifies a core molecular pathway that regulates fibrosis in several organs

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl(4)-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases

Quantitative temporal viromics: an approach to investigate host-pathogen interaction

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called “quantitative temporal viromics” (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model

Studies on CAR and CLMP, two proteins of epithelial tight junctions

Tight junctions are important structures for the function of epithelial cells. They are composed of multi-protein complexes that connect the plasma membranes of two adjacent epithelial cells to each other and function as paracellular barriers, which regulate flux of ions, solutes, proteins and cells across the epithelium. Classical transmembrane components of tight junctions include the tetra- membrane- spann i ng proteins claudins and occludin, which are involved in the formation of these junction complexes. In this thesis, a newly identified group of tight junction proteins is described. These proteins belong to the cortical thymocyte marker in Xenopus (CTX)-subfamily, which belongs to the larger immunoglobulin superfamily of cell adhesion molecules. All CTX-like proteins are type-1 single spanning transmembrane proteins that are composed of two extracellular immunoglobulin loops and an intracellular tail containing a PDZ-domain binding motif, which is used for interactions with PDZ-domain containing proteins that serve as scaffolds, anchoring the transmembrane components of tight junctions to the cytoskeleton. CTX-like proteins mediate cell-cell adhesion and have the capacity to increase transepithelial electrical resistance (TER) across epithelial cells, which suggests a role in barrier function. One member of the CTX-family has been of particular interest: the coxsackie and adenovirus receptor (CAR). CAR was originally identified as a virus receptor and has, as such, been in focus in various adenovirus-based gene transfer and gene therapy studies. Its in vitro properties as being a cell adhesion molecule, expressed at epithelial tight junctions, have also been analyzed. The physiological role of CAR in vivo, however, is less clear. The major aims of these studies were to determine the function of CAR and a newly identified member of the CTX-family, CAR-like membrane protein (CLMP), in adult tissues and during embryonic development. The ligand-of-numb protein-X (LNX) was identified as a novel interacting partner of CAR and co-localized with CAR at cell-cell contacts in epithelial cells. Moreover, CAR was essential for the recruitment of LNX to this sub-cellular localization. In the adult mouse, CAR was predominantly expressed in epithelial cells, where it localized to tight junctions. The intracellular tail of CAR was essential for correct tight junction localization in vivo. A positive correlation between CAR expression and tight junction maturity was found in epithelial cells lining different segments of the tubules in the kidneys. This suggested a role for CAR in regulating permeability and tissue homeostasis, which was further supported by results from studies of the effect of CAR knockdown during zebrafish development. Fish embryos lacking CAR expression in all cells and tissues developed abnormalities that were associated with kidney failure, such as pericardial and body edema and formation of renal cysts. These studies showed that CAR is essential for proper kidney development and function and thus uncovered a novel function for CAR, which had not been detected in mouse embryos lacking CAR expression. CLMP, which was identified through computer-based database searches, was similarly to CAR, expressed in epithelial cells, where it localized to tight junctions. Functional studies showed that CLMP could mediate cell-cell adhesion and increase TER across epithelial cells in culture. In conclusion, the findings presented in this thesis expand our knowledge of the physiological function of CAR and give new insights into the complexity of the CTX-subfamily group of proteins