-
The Journal of Physiology Mar 2018Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these...
KEY POINTS
Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these transmural gradients is proportional to the local resistance. The greatest resistance to oxygen transport into skeletal muscle is considered to reside in the short distance between red blood cells and myocytes. Although crucial to oxygen transport, little is known about transmural pressure gradients within skeletal muscle during contractions. We evaluated oxygen pressures within both the skeletal muscle microvascular and interstitial spaces to determine transmural gradients during the rest-contraction transient in anaesthetized rats. The significant transmural gradient observed at rest was sustained during submaximal muscle contractions. Our findings support that the blood-myocyte interface provides substantial resistance to oxygen diffusion at rest and during contractions and suggest that modulations in microvascular haemodynamics and red blood cell distribution constitute primary mechanisms driving increased transmural oxygen flux with contractions.
ABSTRACT
Oxygen pressure (PO2) gradients across the blood-myocyte interface are required for diffusive O transport, thereby supporting oxidative metabolism. The greatest resistance to O flux into skeletal muscle is considered to reside between the erythrocyte surface and adjacent sarcolemma, although this has not been measured during contractions. We tested the hypothesis that O gradients between skeletal muscle microvascular (PO2 mv ) and interstitial (PO2 is ) spaces would be present at rest and maintained or increased during contractions. PO2 mv and PO2 is were determined via phosphorescence quenching (Oxyphor probes G2 and G4, respectively) in the exposed rat spinotrapezius during the rest-contraction transient (1 Hz, 6 V; n = 8). PO2 mv was higher than PO2 is in all instances from rest (34.9 ± 6.0 versus 15.7 ± 6.4) to contractions (28.4 ± 5.3 versus 10.6 ± 5.2 mmHg, respectively) such that the mean PO2 gradient throughout the transient was 16.9 ± 6.6 mmHg (P < 0.05 for all). No differences in the amplitude of PO2 fall with contractions were observed between the microvasculature and interstitium (10.9 ± 2.3 versus 9.0 ± 3.5 mmHg, respectively; P > 0.05). However, the speed of the PO2 is fall during contractions was slower than that of PO2 mv (time constant: 12.8 ± 4.7 versus 9.0 ± 5.1 s, respectively; P < 0.05). Consistent with our hypothesis, a significant transmural gradient was sustained (but not increased) from rest to contractions. This supports that the blood-myocyte interface is the site of a substantial PO2 gradient driving O diffusion during metabolic transients. Based on Fick's law, elevated O flux with contractions must thus rely primarily on modulations in effective diffusing capacity (mainly erythrocyte haemodynamics and distribution) as the PO2 gradient is not increased.
Topics: Animals; Male; Microvessels; Muscle Cells; Muscle Contraction; Muscle, Skeletal; Oxygen; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Rest
PubMed: 29288568
DOI: 10.1113/JP275170 -
Biochemical and Biophysical Research... May 2019Vascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow....
Vascular pericytes and smooth muscle cells surround many blood vessels of the body. Their primary roles include vessel stabilization and regulation of the blood flow. The high degree of heterogeneity among these cells is dictated by (1) differences in their developmental origin and (2) their location in the vascular bed. Phenotype switching contributes to this heterogeneity especially following in vitro culture. In the absence of distinguishing molecular markers, functional assays that capture their heterogeneity in vitro are needed. Spatiotemporal changes in intracellular Ca levels and contraction in response to vasoconstrictors reflect the differences between vascular pericyte and smooth muscle cell. In order to capture this heterogeneity in vitro, large ensembles of cells need to be analyzed. Here we developed an automated image processing method to measure intracellular Ca and contraction in large cell groups which in combination with a computational approach for integrative analysis allowed vascular pericytes and smooth muscle cells to be distinguished without knowledge of their anatomical origin.
Topics: Calcium Signaling; Cell Line; Equipment Design; Humans; Image Processing, Computer-Assisted; Lab-On-A-Chip Devices; Microscopy, Confocal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Optical Imaging; Pericytes
PubMed: 30940350
DOI: 10.1016/j.bbrc.2019.03.143 -
The Journal of Biological Chemistry Mar 2018Restenosis arises after vascular injury and is characterized by arterial wall thickening and decreased arterial lumen space. Vascular injury induces the production of...
Restenosis arises after vascular injury and is characterized by arterial wall thickening and decreased arterial lumen space. Vascular injury induces the production of thrombin, which in addition to its role in blood clotting acts as a mitogenic and chemotactic factor. In exploring the molecular mechanisms underlying restenosis, here we identified (LIM and cysteine-rich domains 1) as a gene highly responsive to thrombin in human aortic smooth muscle cells (HASMCs). Of note, LMCD1 depletion inhibited proliferation of human but not murine vascular smooth muscle cells. We also found that by physically interacting with E2F transcription factor 1, LMCD1 mediates thrombin-induced expression of the CDC6 (cell division cycle 6) gene in the stimulation of HASMC proliferation. Thrombin-induced LMCD1 and CDC6 expression exhibited a requirement for protease-activated receptor 1-mediated Gα-dependent activation of phospholipase C β3. Moreover, the expression of LMCD1 was highly induced in smooth muscle cells located at human atherosclerotic lesions and correlated with CDC6 expression and that of the proliferation marker Ki67. Furthermore, the LMCD1- and SMCαactin-positive cells had higher cholesterol levels in the atherosclerotic lesions. In conclusion, these findings indicate that by acting as a co-activator with E2F transcription factor 1 in CDC6 expression, LMCD1 stimulates HASMC proliferation and thereby promotes human atherogenesis, suggesting an involvement of LMCD1 in restenosis.
Topics: Adult; Aged; Animals; Atherosclerosis; Co-Repressor Proteins; Female; Humans; LIM Domain Proteins; Male; Mice; Middle Aged; Myocytes, Smooth Muscle; Rats; Thrombin; Young Adult
PubMed: 29326163
DOI: 10.1074/jbc.RA117.000866 -
Circulation Research Mar 2021The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to... (Review)
Review
The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.
Topics: Animals; Cell Differentiation; Cellular Reprogramming Techniques; Humans; Induced Pluripotent Stem Cells; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Signal Transduction
PubMed: 33818124
DOI: 10.1161/CIRCRESAHA.120.318049 -
Arteriosclerosis, Thrombosis, and... Dec 2021
Topics: Aorta; Elastin; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima
PubMed: 34706558
DOI: 10.1161/ATVBAHA.121.317021 -
ACS Nano Jul 2015Transcription factors (TFs) are multidomain proteins that play a critical role in orchestrating stem cell differentiation, but several limitations hinder the full...
Transcription factors (TFs) are multidomain proteins that play a critical role in orchestrating stem cell differentiation, but several limitations hinder the full potential of TF-based gene regulation. Here we report a unique strategy to emulate TFs and differentiate stem cells in a nonviral approach using an artificial, nanoparticle-based transcription factor called NanoScript. The NanoScript platform consists of a gold nanoparticle functionalized with small molecules that mimic the various domains of TFs. As a result, NanoScript mimics the function and structure of TF proteins. Specifically, NanoScript was designed to regulate muscle cell differentiation by targeting myogenic regulatory factors (MRFs), which play an important role in inducing myogenesis. This NanoScript-MRF is stable in physiological environments, localizes within the nucleus, induces differentiation of adipose-derived mesenchymal stem cells into mature muscle cells in 7 days, and is naturally excreted from induced muscle cells. As such, NanoScript represents a safe and powerful tool for applications requiring gene manipulation.
Topics: Cell Differentiation; Cell Line; Gold; Humans; Mesenchymal Stem Cells; Metal Nanoparticles; Muscle Cells; Muscle Development; Myogenic Regulatory Factors
PubMed: 26108385
DOI: 10.1021/acsnano.5b00709 -
The European Respiratory Journal Jan 2020The lung mesenchyme gives rise to multiple distinct lineages of cells in the mature respiratory system, including smooth muscle cells of the airway and vasculature....
RATIONALE
The lung mesenchyme gives rise to multiple distinct lineages of cells in the mature respiratory system, including smooth muscle cells of the airway and vasculature. However, a thorough understanding of the specification and mesenchymal cell diversity in the human lung is lacking.
METHODS
We completed single-cell RNA sequencing analysis of fetal human lung tissues. Canonical correlation analysis, clustering, cluster marker gene identification and t-distributed stochastic neighbour embedding representation was performed in Seurat. Cell populations were annotated using ToppFun. Immunohistochemistry and hybridisation were used to validate spatiotemporal gene expression patterns for key marker genes.
RESULTS
We identified molecularly distinct populations representing "committed" fetal human lung endothelial cells, pericytes and smooth muscle cells. Early endothelial lineages expressed "classic" endothelial cell markers (platelet endothelial cell adhesion molecule/CD31 and claudin 5), while pericytes expressed platelet-derived growth factor receptor-β, Thy-1 membrane glycoprotein and basement membrane molecules (collagen IV, laminin and proteoglycans). We observed a large population of "nonspecific" human lung mesenchymal progenitor cells characterised by expression of collagen I and multiple elastin fibre genes (, and ). We closely characterised the diversity of mesenchymal lineages defined by α-smooth muscle actin () expression. Two cell populations, with the highest levels of transcriptional activity, expressed unique sets of markers associated with airway or vascular smooth muscle cells. Spatiotemporal analysis of these marker genes confirmed early and persistent spatial specification of airway (, and ) and vascular ( and ) smooth muscle cells in the developing human lung.
CONCLUSION
Our data suggest that specification of distinct airway and vascular smooth muscle cell phenotypes is established early in development and can be identified using the markers we provide.
Topics: Cell Differentiation; Cell Lineage; Endothelial Cells; Humans; Lung; Mesenchymal Stem Cells; Myocytes, Smooth Muscle
PubMed: 31619469
DOI: 10.1183/13993003.00746-2019 -
Cytoskeleton (Hoboken, N.J.) Mar 2013Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle...
Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized in this region. Additionally, we identified a classical DNA-binding leucine zipper domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA-binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis.
Topics: Animals; Annexin A5; Apoptosis; Cell Line; Cell Nucleus; Flow Cytometry; Leucine Zippers; Mice; Muscle Cells; Mutagenesis, Site-Directed; Nuclear Localization Signals; Troponin T
PubMed: 23378072
DOI: 10.1002/cm.21095 -
BMC Biology Apr 2016Skeletal muscle stem cells enable the formation, growth, maintenance, and regeneration of skeletal muscle throughout life. The regeneration process is compromised in...
BACKGROUND
Skeletal muscle stem cells enable the formation, growth, maintenance, and regeneration of skeletal muscle throughout life. The regeneration process is compromised in several pathological conditions, and muscle progenitors derived from pluripotent stem cells have been suggested as a potential therapeutic source for tissue replacement. DNA methylation is an important epigenetic mechanism in the setting and maintenance of cellular identity, but its role in stem cell determination towards the myogenic lineage is unknown. Here we addressed the DNA methylation dynamics of the major genes orchestrating the myogenic determination and differentiation programs in embryonic stem (ES) cells, their Pax7-induced myogenic derivatives, and muscle stem cells in proliferating and differentiating conditions.
RESULTS
Our data showed a common muscle-specific DNA demethylation signature required to acquire and maintain the muscle-cell identity. This specific-DNA demethylation is Pax7-mediated, and it is a prime event in muscle stem cells gene activation. Notably, downregulation of the demethylation-related enzyme Apobec2 in ES-derived myogenic precursors reduced myogenin-associated DNA demethylation and dramatically impaired the expression of differentiation markers and, ultimately, muscle differentiation.
CONCLUSIONS
Our results underscore DNA demethylation as a key mechanism driving myogenesis and identify specific Pax7-mediated DNA demethylation signatures to acquire and maintain the muscle-cell identity. Additionally, we provide a panel of epigenetic markers for the efficient and safe generation of ES- and induced pluripotent stem cell (iPS)-derived myogenic progenitors for therapeutic applications.
Topics: Animals; Cell Differentiation; Cell Line; Cells, Cultured; CpG Islands; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Humans; Mice; Muscle Cells; Muscle Development; PAX7 Transcription Factor; Promoter Regions, Genetic
PubMed: 27075038
DOI: 10.1186/s12915-016-0250-9 -
Microcirculation (New York, N.Y. : 1994) May 2013Dynamic changes in intracellular Ca²⁺ levels in vascular smooth muscle cells are critically important for cardiovascular regulation. This Special Topic Issue...
Dynamic changes in intracellular Ca²⁺ levels in vascular smooth muscle cells are critically important for cardiovascular regulation. This Special Topic Issue highlights a series of expert opinion articles focused on this important subject. After a brief overview, novel discoveries surrounding smooth muscle cell Ca²⁺ influx via L-type and T-type channels are reviewed. Current work revealing the functional importance of dynamic Ca²⁺ signaling in the control of the parenchymal microvasculature and the emerging role of mitochondrial Ca²⁺ signaling and store-operated Ca²⁺ entry in smooth muscle cells is discussed. Finally, recent data describing a new target of localized Ca²⁺ signaling in arterial myocytes that is responsible for membrane depolarization is reviewed. Authors were encouraged to write in an opinionated and provocative manner with the hope of stimulating discussion in this area of research.
Topics: Animals; Calcium; Calcium Channels, L-Type; Calcium Channels, T-Type; Calcium Signaling; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle
PubMed: 23421765
DOI: 10.1111/micc.12049