A multi-stem cell basis for craniosynostosis and calvarial mineralization.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-ß1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.

Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II-stimulated cardiac fibroblasts.

Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-ß1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-ß1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-ß1 (TGF-ß1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-ß1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-ß1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-ß1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-ß1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.

Ascorbate starvation alters endoplasmic reticulum-resident enzymes in cardiac fibroblasts, priming them for increased procollagen secretion.

Since ascorbate is unnecessary for cell growth and survival, cardiac fibroblasts are routinely cultured without it. However, ascorbate is necessary for optimal collagen synthesis, so we hypothesized that its presence would influence cell phenotype. Cardiac fibroblasts cultured without ascorbate had increased intracellular levels of procollagens, with procollagen α1(III) showing the largest accumulation. Endoplasmic reticulum (ER)-resident proteins that are known to bind single-stranded procollagens were also elevated. These included the catalytic prolyl 4-hydroxylase subunits, lysyl hydroxylases, and hydroxylysyl galactosyltransferases, with prolyl 4-hydroxylase α1 and α2 (P4HA1 and P4HA2) demonstrating the largest increases. There were no differences in the levels of protein disulfide isomerase (P4HB/PDI) or the triple-helical procollagen chaperone, HSP47, with or without ascorbate. Results were similar with mouse and rat cardiac fibroblasts, suggesting a conserved response. Ascorbate-replete cells that were subsequently deprived of the vitamin lost the ability to secrete intact procollagen α1(I) within ~3days, approximately when intracellular procollagen α1(III) and P4HA1 levels began to rise. Upon ascorbate re-addition, starved fibroblasts initially secreted high levels of procollagen that gradually declined over ~4days, a pattern that was not universal as extra domain A (EDA)-fibronectin secretion was unchanged. Despite the necessity of the P4HA enzymes for triple-helical procollagen formation, they were not responsible for early increased secretion. However, in the absence of ascorbate, P4HA2 overexpression increased intracellular turnover of procollagens, suggesting that it may help clear accumulating procollagens from the ER. Cardiac fibroblasts change in the absence of ascorbate to cope with increased intracellular levels of procollagens. These changes occur slowly and can render the cells phenotypically altered for several days after ascorbate re-addition. These findings have direct implications for the study of cardiac fibroblasts in culture, and may help our understanding of the response of these cells to fluctuating nutrient levels in ischemic myocardium.

Discoidin domain receptor 2 germline gene deletion leads to altered heart structure and function in the mouse.

Discoidin domain receptor 2 (DDR2) is a fibrillar collagen receptor that is expressed in mesenchymal cells throughout the body. In the heart, DDR2 is selectively expressed on cardiac fibroblasts. We generated a germline DDR2 knockout mouse and used this mouse to examine the role of DDR2 deletion on heart structure and function. Echocardiographic measurements from null mice were consistent with those from a smaller heart, with reduced left ventricular chamber dimensions and little change in wall thickness. Fractional shortening appeared normal. Left ventricular pressure measurements revealed mild inotropic and lusitropic abnormalities that were accentuated by dobutamine infusion. Both body and heart weights from 10-wk-old male mice were ~20% smaller in null mice. The reduced heart size was not simply due to reduced body weight, since cardiomyocyte lengths were atypically shorter in null mice. Although normalized cardiac collagen mass (assayed by hydroxyproline content) was not different in null mice, the collagen area fraction was statistically higher, suggesting a reduced collagen density from altered collagen deposition and cross-linking. Cultured cardiac fibroblasts from null mice deposited collagen at a slower rate than wild-type littermates, possibly due to the expression of lower prolyl 4-hydroxylase α-isoform 1 enzyme levels. We conclude that genetic deletion of the DDR2 collagen receptor alters cardiac fibroblast function. The resulting perturbations in collagen deposition can influence the structure and function of mature cardiomyocytes.

Control of craniofacial development by the collagen receptor, discoidin domain receptor 2.

Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene (DDR2), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.

We each have unique facial features that are key to our identities. These features are inherited, but the mechanisms are poorly understood. People with the genetic disease spondylo-meta-epiphyseal dysplasia, or SMED, have characteristic facial and skull abnormalities including a flattened face and shortened skull. SMED is associated with mutations that inactivate the gene encoding a protein called discoidin domain receptor 2 (DDR2), which is a receptor for collagen. Collagen is the major structural protein in the human body, supporting the structure of cells and tissues. It also controls cell behaviors including growth, migration and differentiation, and it helps form tissues such as cartilage or bone. At least some of the effects of collagen on cells depend on its interaction with DDR2. Since the facial and skull abnormalities in mice with mutations that stop DDR2 from working correctly resemble those of SMED patients, these mice can be used to understand the cellular basis for this disease, as well as the role of DDR2 in the embryonic development of the face and skull. Therefore, Mohamed et al. set out to understand how loss of DDR2 causes the characteristic facial and skull defects associated with SMED. Mohamed et al. used mice that had been genetically modified so that DDR2 could be inactivated in skeletal progenitor cells, cartilage cells and bone cells (osteoblasts). Examining these mice, they found that the shortened skulls and flat face characteristic of mice lacking DDR2 are due to bones at the skull base failing to elongate correctly due to defects in the growth centers that depend on cartilage. Mohamed et al. also discovered that the cells that normally produce DDR2 are the progenitors of cartilage and bone-forming cells, which partly explains why lacking this protein leads to issues in growth of these tissues. In addition to shedding light on the causes of SMED, Mohamed et al.'s results also provide general insights into the mechanisms controlling the formation of facial and skull bones that depend on interactions between cells and collagen. This information may help explain how other abnormalities in the face and skull emerge, and provide a basis for how the shape of the skull has changed during human evolution. In the future, it may be possible to manipulate the activity of DDR2 to correct skull defects.

The collagen receptor, discoidin domain receptor 2, functions in Gli1-positive skeletal progenitors and chondrocytes to control bone development.

Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor kinase that, together with integrins, is required for cells to respond to the extracellular matrix. Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development. However, the cellular functions of Ddr2 in bone are not understood. Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum. Consistent with these findings, Ddr2+ cells could differentiate into hypertrophic chondrocytes, osteoblasts, and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker, Gli1. A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts. Furthermore, Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation, respectively. This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.

Targeting the ACE2-Ang-(1-7) pathway in cardiac fibroblasts to treat cardiac remodeling and heart failure.

Fibroblasts play a pivotal role in cardiac remodeling and the development of heart failure through the deposition of extra-cellular matrix (ECM) proteins and also by affecting cardiomyocyte growth and function. The renin-angiotensin system (RAS) is a key regulator of the cardiovascular system in health and disease and many of its effects involve cardiac fibroblasts. Levels of angiotensin II (Ang II), the main effector molecule of the RAS, are elevated in the failing heart and there is a substantial body of evidence indicating that this peptide contributes to changes in cardiac structure and function which ultimately lead to progressive worsening in heart failure. A pathway involving angiotensin converting enzyme 2 (ACE2) has the capacity to break down Ang II while generating angiotensin-(1-7) (Ang-(1-7)), a heptapeptide, which in contrast to Ang II, has cardioprotective and anti-remodeling effects. Many Ang-(1-7) actions involve cardiac fibroblasts and there is information indicating that it reduces collagen production and also may protect against cardiac hypertrophy. This report describes the effects of ACE2 and Ang-(1-7) that appear to be relevant in cardiac remodeling and heart failure and explores potential therapeutic strategies designed to increase ACE2 activity and Ang-(1-7) levels to treat these conditions. This article is part of a special issue entitled ''Key Signaling Molecules in Hypertrophy and Heart Failure.''

Effects of ACE2 inhibition in the post-myocardial infarction heart.

BACKGROUND: There is evidence that angiotensin-converting enzyme 2 (ACE2) is cardioprotective. To assess this in the post-myocardial infarction (MI) heart, we treated adult male Sprague-Dawley rats with either placebo (PL) or C16, a selective ACE2 inhibitor, after permanent coronary artery ligation or sham operation. METHODS AND RESULTS: Coronary artery ligation resulting in MI between 25% to 50% of the left ventricular (LV) circumference caused substantial cardiac remodeling. Daily C16 administration from postoperative days 2 to 28 at a dose that inhibited myocardial ACE2 activity was associated with a significant increase in MI size and reduction in LV % fractional shortening. Treatment with C16 did not significantly affect post-MI increases in LV end-diastolic dimension but did inhibit increases in wall thickness and fibrosis in non-infarcted LV. On postoperative day 7, C16 had no significant effect on the increased level of apoptosis in the infarct and border zones nor did it significantly affect capillary density surrounding the MI. It did, however, significantly reduce the number of c-kit(+) cells in the border region. CONCLUSIONS: These findings support the notion that ACE2 exerts cardioprotective effects by preserving jeopardized cardiomyocytes in the border zone. The reduction in hypertrophy and fibrosis with C16, however, suggests that ACE2 activity has diverse effects on post-MI remodeling.

Mechanisms of cardiac collagen deposition in experimental models and human disease.

The inappropriate deposition of extracellular matrix within the heart (termed cardiac fibrosis) is associated with nearly all types of heart disease, including ischemic, hypertensive, diabetic, and valvular. This alteration in the composition of the myocardium can physically limit cardiomyocyte contractility and relaxation, impede electrical conductivity, and hamper regional nutrient diffusion. Fibrosis can be grossly divided into 2 types, namely reparative (where collagen deposition replaces damaged myocardium) and reactive (where typically diffuse collagen deposition occurs without myocardial damage). Despite the widespread association of fibrosis with heart disease and general understanding of its negative impact on heart physiology, it is still not clear when collagen deposition becomes pathologic and translates into disease symptoms. In this review, we have summarized the current knowledge of cardiac fibrosis in human patients and experimental animal models, discussing the mechanisms that have been deduced from the latter in relation to the former. Because assessment of the extent of fibrosis is paramount both as a research tool to further understanding and as a clinical tool to assess patients, we have also summarized the current state of noninvasive/minimally invasive detection systems for cardiac fibrosis. Albeit not exhaustive, our aim is to provide an overview of the current understanding of cardiac fibrosis, both clinically and experimentally.

Improving the titer of recombinant adenovirus by suppressing problematic transgene transcription during packaging.

A new strategy to package "problematic" transgenes in adenovirus was developed that was based on modifications of the tetracycline-inducible system. This strategy used two components: the adenoviral genome containing the transgene under control of a hybrid TRE promoter/SV40 enhancer and a trans-encoded tTS suppressor Using luciferase reporters, expression of tTS in 293A cells reduced transcription from the promoter/enhancer 25-fold. Procaspase 8 adenovirus was then tested, since it is known to package poorly with standard adenoviral systems. Expression of tTS in 293A cells increased the titer of procaspase 8 adenovirus by 22-fold in initial viral packaging (using transiently transfected tTS) and 9-fold in subsequent viral reamplification (using 293A cells stably expressing tTS). The Tac antigen gene (i.e., CD25), which packages in adenovirus without difficulty, was also tested as a control. In contrast to that observed with procaspase 8, tTS expression did not alter the titer obtained when packaging the CD25 gene, thus excluding nonspecific effects of tTS expression on adenoviral titer Since tTS was provided in trans and did not package in the resulting adenoviruses, strong transcription of the transgenes occurred in transducted cells without the need of additional reagents.

Tumor necrosis factor-alpha-induced AT1 receptor upregulation enhances angiotensin II-mediated cardiac fibroblast responses that favor fibrosis.

Extracellular matrix (ECM) remodeling after myocardial infarction (MI) is an important determinant of cardiac function. Tumor necrosis factor-alpha (TNF-alpha) and angiotensin (Ang) II levels increase after MI and both factors affect fibroblast functions. The type 1 (AT1) receptor that mediates most Ang II effects is upregulated after MI in cardiac fibroblasts, and there is evidence that this is caused by TNF-alpha. We sought to determine if TNF-alpha-induced AT1 receptor upregulation alters fibroblast responsiveness to Ang II and if this effect differs from direct TNF-alpha effects on fibroblast functions. In cultured neonatal rat cardiac fibroblasts, TNF-alpha reduced cellular [3H]-proline incorporation, increased matrix metalloproteinase-2 (MMP-2) activity and protein, and increased TIMP-1 protein levels. In cardiac fibroblasts with TNF-alpha-induced AT1 receptor upregulation, Ang II-stimulated [3H]proline incorporation and TIMP-1 protein production was approximately 2-fold greater than in nonpretreated fibroblasts. Angiotensin II reduced MMP-2 activity and protein level only in TNF-alpha-pretreated fibroblasts. Angiotensin II effects were inhibited by selective AT1 (but not AT2) receptor blockers. Thus, TNF-alpha-induced AT1 receptor upregulation enhances Ang II-mediated functions that favor fibrosis. These effects are mostly directionally opposite of direct TNF-alpha effects on cardiac fibroblasts. Recognition of multifaceted TNF-alpha effects provides new insights into post-MI ECM remodeling.

IL-1beta and TNF-alpha upregulate angiotensin II type 1 (AT1) receptors on cardiac fibroblasts and are associated with increased AT1 density in the post-MI heart.

Angiotensin (Ang) II plays an important role in post-myocardial infarction (MI) cardiac remodeling. The Ang II type 1 (AT(1)) receptor which mediates most Ang II effects is upregulated on non-myocytes in the post-MI heart. We have shown that pro-inflammatory cytokines increase AT(1) receptor density on cardiac fibroblasts through a mechanism involving NF-kappaB activation. This study examines the in vitro kinetics of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced AT(1) receptor upregulation in neonatal rat cardiac fibroblasts and assesses temporal and spatial associations between the appearance of these agents and increased AT(1) receptor density post-MI. The results show that IL-1beta more rapidly induces AT(1) receptor upregulation than does TNF-alpha, an effect that can be mimicked by a NF-kappaB-dependent luciferase reporter gene. Moreover, the effects of these pro-inflammatory cytokines are additive. Using immunohistochemistry in the post-MI rat heart we found strong temporal and spatial correlations between TNF-alpha, IL-1beta and AT(1) receptor proteins in the peri-infarction (PI) zone in fibroblasts and macrophages. Labeling intensity for the cytokines and the AT(1) receptor increased from 1 to 7 days post-MI in the PI zone in conjunction with replacement scar formation. This labeling persisted in non-myocytes bordering the scar for up to 83 days post-MI. These findings suggest that IL-1beta and TNF-alpha act coordinately to increase AT(1) receptor density on non-myocytes in the post-MI heart and that this effect may contribute to extracellular matrix remodeling and fibrosis.

Effects of cytokine treatment on angiotensin II type 1A receptor transcription and splicing in rat cardiac fibroblasts.

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (AT(1A)) receptor. The cytokines tumor necrosis factor-alpha and interleukin-1beta (IL-1beta) were shown previously to upregulate AT(1A) receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-kappaB (NF-kappaB)-dependent transcription and also, to a lesser extent, altered mRNA splicing in the mechanism of receptor upregulation. Cytokine stimulation was found to increase AT(1A) heterogeneous nuclear RNA levels, which strongly suggests that mRNA upregulation occurs transcriptionally. The transcription factor NF-kappaB was previously deemed necessary for cytokine-induced AT(1A) receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-kappaB elements at -365 and -2540 bp. Both isolated elements were shown to bind NF-kappaB (using gel-shift assays) and to transactivate a minimal promoter (using reporter assays), although the element at -365 bp appeared stronger. Three splice variants of AT(1A) receptor mRNA that have different 5' untranslated regions were detected in rat tissues, namely, exons 1-2-3 (predominant), 1-2-3+6, and 1-3. Cytokine treatment of fibroblasts upregulated all splice variants, but exon 1-3 increased more than the others. This differential upregulation, albeit of modest magnitude, was statistically significant with IL-1beta treatment. Exon 2 contains an inhibitory minicistron and a predicted inhibitory hairpin structure. Luciferase reporter assays indicated that each splice variant translates at a different efficiency, with exon 1-2-3+6 (both minicistron and hairpin) < exon 1-2-3 (minicistron only) < exon 1-3 (neither minicistron or hairpin). These results provide evidence that cytokines increase AT(1) protein levels by altering both transcription and splicing.