Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.
In contrast to cold blooded vertebrates, the ability to regenerate morphologically and functionally complex structures is limited in adult mammals. Recruitment of progenitor cells is a key step in the regenerative process. The possibility of repairing missing or diseased tissues in humans has been potentiated by the increasing understanding of somatic stem cells, their plasticity and the possibility of modulating it, that could be harnessed either to stimulate endogenous repair or to engineer the required tissue. Here, we focus on human mesenchymal stem cells (MSCs), important players in tissue homeostasis in healthy organisms, with a particular emphasis on those derived from the adipose tissue (ADSCs). While a mark of MSC identity is the ability to differentiate into osteoblasts, chondrocytes and adipocytes, there is evidence that their potential goes beyond these three mesenchymal lineages. We discuss some differentiation and modulatory properties of MSCs and provide an overview of our recent work on ADSCs from paediatric patients (pADSCs) that has shown their ability to give raise to non-mesenchymal cells, consistent with a significant plasticity. Finally, we present novel data indicating that both mesenchymal lineages (adipogenic, chondrogenic and osteogenic) and neural and epithelial lineages can originate from clonal lines that like the parental line express markers of pluripotency as well as the stromal cell marker, GREM1. Together these data support the existence of pADSC multipotent stem cells.
The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. this observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αc-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation. The 61 human AGC kinases form a monophyletic group of serine/threonine kinases that preferably phosphorylates residues in close proximity of basic amino acids such as Arg (R) and Lys (K) 1,2. The kinase domains (KD) of all the AGC kinases share the same tertiary structure, characterized by an amino-terminal small lobe (N-lobe) and a carboxy-terminal large lobe (C-lobe), as originally described for PKA 3. The two lobes form a pocket that binds one molecule of ATP as phosphate donor during substrate phosphorylation. The transition from inactive to active state in AGC kinases is achieved through conformational rearrangements of key structural elements, such as the activation segment and the αC-helix. The activation segment is a sequence of variable length (from 25 aa of PKAa to 43 aa of MAST1) spanning from Asp-Phe-Gly (DFG) to Ala-Pro-Glu (APE) sequences, and including the activation loop (AL) and the P + 1 loop 4. The DFG sequence is part of the ATP binding site whose orientation defines the active (DFG-in) 3 and the inactive (DFG-out) states of AGC kinases 5. The AL contains, in 43 out of 61 AGC kinases (Fig. 1A), a key phosphorylatable site (consensus sequence S/T-x-x-G-T), found to be substrate of 3-Phosphoinositide-dependent protein kinase-1 (PDK1) 6. The phosphate group added on the AL form a complex set of salt bridges with basic amino acid groups, that in PKA are respectively: R165 in the catalytic loop, H87 in the αC-helix and K189 in the AL, just after the DFG motif 7,8. By connecting these residues, the phosphorylation of the AL promotes the transition in a more ordered conformation, the stabilization of the two lobes in the closed/active conformation and the assembly of a key hydrophobic core, defined R-spine 9-11. Crucial event in the tr...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.