A three-dimensional model with two-body interactions for endothelial cells in angiogenesis.

We introduce a three-dimensional mathematical model for the dynamics of vascular endothelial cells during sprouting angiogenesis. Angiogenesis is the biological process by which new blood vessels form from existing ones. It has been the subject of numerous theoretical models. These models have successfully replicated various aspects of angiogenesis. Recent studies using particle-based models have highlighted the significant influence of cell shape on network formation, with elongated cells contributing to the formation of branching structures. While most mathematical models are two-dimensional, we aim to investigate whether ellipsoids also form branch-like structures and how their shape affects the pattern. In our model, the shape of a vascular endothelial cell is represented as a spheroid, and a discrete dynamical system is constructed based on the simple assumption of two-body interactions. Numerical simulations demonstrate that our model reproduces the patterns of elongation and branching observed in the early stages of angiogenesis. We show that the pattern formation of the cell population is strongly dependent on the cell shape. Finally, we demonstrate that our current mathematical model reproduces the cell behaviours, specifically cell-mixing, observed in sprouts.

Persistent homological cell tracking technology.

In this paper, we develop a cell tracking method based on persistent homological figure detection technology. We apply our tracking method to 9 different time-series cell images and extract several kinds of cell movements. Being able to analyze various images with a single method allows researchers to systematically understand and compare different tracking data. Persistent homological cell tracking technology's 9 parameters all have clear meanings. Thus, researchers can decide the parameters not by black box trial-and-error but by the purpose of their analysis. We use model data with ground truth to see our method's performance. We compare persistent homological figure detection and cell tracking technology with Image-Pro, sure-foreground in watershed method, and cell detection methods in previous studies. We see that there are some cases where Image-Pro's tracking stops and requires manual plots, while our method does not require manual plots. We show that our technology includes sure-foreground and has more information, and can be applied to different types of data that previously needed different methods. We also show that our technology is powerful as a detection technology by applying the technology to 5 different types of cell images.

Coordinated linear and rotational movements of endothelial cells compartmentalized by VE-cadherin drive angiogenic sprouting.

Angiogenesis is a sequential process to extend new blood vessels from preexisting ones by sprouting and branching. During angiogenesis, endothelial cells (ECs) exhibit inhomogeneous multicellular behaviors referred to as "cell mixing," in which ECs repetitively exchange their relative positions, but the underlying mechanism remains elusive. Here we identified the coordinated linear and rotational movements potentiated by cell-cell contact as drivers of sprouting angiogenesis using in vitro and in silico approaches. VE-cadherin confers the coordinated linear motility that facilitated forward sprout elongation, although it is dispensable for rotational movement, which was synchronous without VE-cadherin. Mathematical modeling recapitulated the EC motility in the two-cell state and angiogenic morphogenesis with the effects of VE-cadherin-knockout. Finally, we found that VE-cadherin-dependent EC compartmentalization potentiated branch elongations, and confirmed this by mathematical simulation. Collectively, we propose a way to understand angiogenesis, based on unique EC behavioral properties that are partially dependent on VE-cadherin function.

Murine neonatal ketogenesis preserves mitochondrial energetics by preventing protein hyperacetylation.

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing.

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL-derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex-driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor-deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.

Calpain-6 deficiency promotes skeletal muscle development and regeneration.

Calpains are Ca(2+)-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.

Calpain-6, a microtubule-stabilizing protein, regulates Rac1 activity and cell motility through interaction with GEF-H1.

Crosstalk between microtubules and actin filaments is crucial for various cellular functions, including cell migration, spreading and cytokinesis. The Rac1 GTPase plays a key role in such crosstalk at the leading edge of migrating cells in order to promote lamellipodial formation. However, the mechanism underlying the link between microtubules and Rac1 activation remains unclear. Here, we show that calpain-6 (CAPN6), a non-proteolytic calpain with microtubule-binding and -stabilizing activity, might participate in this crosstalk. Small interfering RNA (siRNA)-induced knockdown of Capn6 in NIH 3T3 cells resulted in Rac1 activation, which promoted cell migration, spreading and lamellipodial protrusion. This increase in Rac1 activity was abolished by knockdown of the Rho guanine nucleotide exchange factor GEF-H1 (officially known as Arhgef2). CAPN6 and GEF-H1 colocalized with microtubules and also interacted with each other through specific domains. Upon knockdown of Capn6, GEF-H1 was shown to translocate from microtubules to the lamellipodial region and to interact with Rac1. By contrast, RhoA activity was decreased upon knockdown of Capn6, although low levels of active RhoA or the presence of RhoA molecules appeared to be required for the Capn6-knockdown-induced Rac1 activation. We suggest that CAPN6 acts as a potential regulator of Rac1 activity, through a mechanism involving interaction with GEF-H1, to control lamellipodial formation and cell motility.

Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation.

The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.

Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest.

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3-/- embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

An endothelin-1 switch specifies maxillomandibular identity.

Articulated jaws are highly conserved structures characteristic of gnathostome evolution. Epithelial-mesenchymal interactions within the first pharyngeal arch (PA1) instruct cephalic neural crest cells (CNCCs) to form the different skeletal elements of the jaws. The endothelin-1 (Edn1)/endothelin receptor type-A (Ednra)-->Dlx5/6-->Hand2 signaling pathway is necessary for lower jaw formation. Here, we show that the Edn1 signaling is sufficient for the conversion of the maxillary arch to mandibular identity. Constitutive activation of Ednra induced the transformation of upper jaw, maxillary, structures into lower jaw, mandibular, structures with duplicated Meckel's cartilage and dermatocranial jaws constituted by 4 dentary bones. Misexpression of Hand2 in the Ednra domain caused a similar transformation. Skeletal transformations are accompanied by neuromuscular remodeling. Ednra is expressed by most CNCCs, but its constitutive activation affects predominantly PA1. We conclude that after migration CNCCs are not all equivalent, suggesting that their specification occurs in sequential steps. Also, we show that, within PA1, CNCCs are competent to form both mandibular and maxillary structures and that an Edn1 switch is responsible for the choice of either morphogenetic program.

Calpain 6 is involved in microtubule stabilization and cytoskeletal organization.

The calpains are a family of Ca(2+)-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated alpha-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated alpha-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.

Endothelin-1 regulates the dorsoventral branchial arch patterning in mice.

Endothelin-1 (ET-1), a 21-amino acid peptide secreted by the epithelium and core mesenchyme in the branchial arches as well as vascular endothelium, is involved in craniofacial and cardiovascular development through endothelin receptor type-A (EdnrA) expressed in the neural crest-derived ectomesenchyme. Here we show that ET-1(-/-) mutant mice exhibit a homeotic-like transformation of the lower jaw to an upper jaw. Most of the maxillary arch-derived components are duplicated and replaced mandibular arch-derived structures, resulting in a mirror image of the upper and lower jaws in the ET-1(-/-) mutant. As for hyoid arch-derivatives, the ventral structures are severely affected in comparison to the dorsal ones in the ET-1(-/-) mutant. Correspondingly, the expression of Dlx5 and Dlx6, Distalless-related homeobox genes determining the ventral identity of the anterior branchial arches, and of the mandibular marker gene Pitx1 is significantly downregulated in the ET-1(-/-) mutant, whereas the expression of Dlx2 and the maxillary marker gene Prx2 is unaffected or rather upregulated. These findings indicate that the ET-1/EdnrA signaling may contribute to the dorsoventral axis patterning of the branchial arch system as a mediator of the regional intercellular interactions.

Staurosporine enhances the expression of tissue inhibitor of metalloproteinase-1 in human prostate cancer cells.

We reported previously that human prostate cancer cell line TSU-Pr1 can differentiate into neuronal cells by staurosporine treatment. In this process, reduction of invasive abilities was observed in staurosporine treated TSU-Pr1 cells. In the present study, we investigated the effect of staurosporine on tissue inhibitor of metalloproteinases (TIMPs) in prostate cancer cells. We show that treatment of TSU-Pr1 cells with staurosporine results in induction of TIMP-1 mRNA and protein secretion. The induction of TIMP-1 mRNA expression by staurosporine is likely to be caused by increased transcriptional activity and this mechanism is indirect. Furthermore, recombinant human TIMP-1 reduces the invasive activity of TSU-Pr1 cells. We are the first to report that mRNA expression and protein secretion of TIMP-1 are enhanced by staurosporine treatment in prostate cancer cells. These findings suggest that enhancement of TIMP-1 is associated with suppression of invasive activity caused by staurosporine treatment.