David J. Pennisi

Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.

The regulation of growth factor localization, availability and activity is critical during embryogenesis to ensure appropriate organogenesis. This process is regulated through the coordinated expression of growth factors and their cognate receptors, as well as via proteins that can bind, sequester or localize growth factors to distinct locations. One such protein is the transmembrane protein Crim1. This protein has been shown to be expressed broadly within the developing embryo, and to regulate organogenesis within the eye, kidney and placenta. Mechanistically, Crim1 has been revealed to mediate organogenesis via its interaction with growth factors including TGF beta s, BMPs, VEGFs and PDFGs. More recently, Crim1 has been shown to influence cardiac development, providing further insights into the function of this protein. This review will provide an overview of the role of Crim1 in organogenesis, largely focusing on how this protein regulates growth factor signaling in the nascent heart. Moreover, we will address the challenges ahead relating to further elucidating how Crim1 functions during development.

Coronary heart disease remains a leading cause of morbidity and mortality, and as such, there is a pressing need to develop efficacious therapies. Any future cell-based therapeutic interventions aimed at revascularization of an ischemic heart, or regeneration of damaged coronary vessels, require a clear understanding of the developmental programs that are in place in the embryo. This knowledge will also be necessary to provide the basis for biologically engineered heart tissues..

The epicardium has a critical role during embryonic development, contributing epicardium-derived lineages to the heart, as well as providing regulatory and trophic signals necessary for myocardial development. Crim1 is a unique trans-membrane protein expressed by epicardial and epicardially-derived cells but its role in cardiogenesis is unknown. Using knockout mouse models, we observe that loss of Crim1 leads to congenital heart defects including epicardial defects and hypoplastic ventricular compact myocardium. Epicardium-restricted deletion of Crim1 results in increased epithelial-to-mesenchymal transition and invasion of the myocardium in vivo, and an increased migration of primary epicardial cells. Furthermore, Crim1 appears to be necessary for the proliferation of epicardium-derived cells (EPDCs) and for their subsequent differentiation into cardiac fibroblasts. It is also required for normal levels of cardiomyocyte proliferation and apoptosis, consistent with a role in regulating epicardium-derived trophic factors that act on the myocardium. Mechanistically, Crim1 may also modulate key developmentally expressed growth factors such as TGFβs, as changes in the downstream effectors phospho-SMAD2 and phospho-ERK1/2 are observed in the absence of Crim1. Collectively, our data demonstrates that Crim1 is essential for cell-autonomous and paracrine aspects of heart development.

Crim1 is a transmembrane protein that regulates the bioavailability of growth factors such as VEGFA. Crim1^{KST264/KST264} hypomorphic mice develop renal disease characterized by glomerular cysts and loss of endothelial integrity, progressing to peritubular and pericystic fibrosis. Peritubular capillary endothelial cells display morphological changes as well as detachment from the basement membrane. In this study, gene expression profiling of CD31⁺ endothelial cells isolated from Crim1^{KST264/KST264} kidneys showed up‐regulation of transcripts associated with fibrosis (Col3a1, Loxl1), endothelial dysfunction (Abp1, Dcn, Lcn2), biomarkers of renal damage (Lcn2, Havcr1/Kim1) as well as evidence for a TGFβ1/TNF‐associated inflammatory process. To determine whether the aberrant endothelium may in part contribute to the fibrogenic process, Tie2Cre‐DsRed lineage tracing was undertaken in Crim1^{KST264/KST264} mice. Approximately 31% of de novo αSMA⁺ myofibroblasts detected within the tubulointerstitium were Tie2⁺DsRed⁺. However, 5.3% were F4/80⁺DsRed⁺, indicating a small population of myofibroblasts of monocytic rather than endothelial origin. In contrast, only 12% of myofibroblasts located around glomerular cysts were Tie2⁺DsRed⁺, with 7.7% being monocyte‐derived (F4/80⁺DsRed⁺). Collectively, this model supports the involvement of endothelial cells/monocytes in fibrosis within the tubulointerstitium, but also the heterogeneity of the fibrotic process even within distinct regions of the same kidney. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Crim1 is a developmentally expressed, transmembrane protein essential for normal embryonic development. We generated mice engineered to contain a Crim1 conditional null allele by flanking exons three and four of Crim1 with unidirectional LoxP sites. After crossing Crim1^+/FLOX mice with a CMV‐Cre line, a Crim1^+/Δflox colony was established after germline transmission of the deleted allele. We then analyzed genomic DNA, mRNA transcripts, and protein expression from Crim1^{Δflox/Δflox} null mice to confirm the nature of the genomic lesion. Crim1^{Δflox/Δflox} mice displayed phenotypes similar to those previously described for a Crim1 gene‐trap mutant, Crim1^{KST264/KST264}, including perinatal lethality, digit syndactyly, eye, and kidney abnormalities, with varying penetrance and severity. The production of a conditional mutant allele represents a valuable resource for the study of the tissue‐specific roles for Crim1, and for understanding the pleimorphic phenotypes associated with Crim1 mutation. genesis 50:711–716, 2012.