Feedback via Ca²⁺-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle

PURPOSE: To investigate the role of feedback by Ca²?-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²?](i) and the amplitude and frequency of Ca²?-waves in retinal arterioles. The Ca²?-activated Cl?-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²?-activated K?-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²?](i). The voltage-operated Ca²?-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²?](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²?-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²?, also require positive feedback via Ca²?-activated Cl?-channels and L-type Ca²?-channels.

Glucose-induced oxidative stress in vascular contractile cells - Comparison of aortic smooth muscle cells and retinal pericytes

Free radical-mediated damage to vascular cells may be involved in the pathogenesis of diabetic vasculopathy. The aim of this study was to compare the extent of glucose-induced oxidative stress in both vascular smooth muscle cells (VSMCs) and pericytes and the effect on antioxidant enzyme gene expression and activities. Porcine aortic VSMC and retinal pericytes were cultured in either 5 or 25 mmol/l glucose for 10 days. Intracellular malondialdehyde (MDA) was measured as a marker of peroxidative damage, and mRNA expression of CuZn-SOD, MnSOD, catalase, and glutathione peroxidase (GPX) were measured by Northern analysis. Glutathione (GSH) was also measured. There was a significant increase in MDA in VSMCs in 25 mmol/l glucose (1.34 +/- 0.11 vs. 1.88 +/- 0.24 nmol/mg protein, 5 vs. 25 mmol/l D-glucose, mean +/- SE, n = 15, P

Pharmacological profiling of store-operated Ca(2+) entry in retinal arteriolar smooth muscle

Objective: Pharmacological profiling of store-operated Ca(2+) entry (SOCE) and molecular profiling of ORAI and TRPC expression in arterioles. 
Methods: Fura-2 based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression were screened using RT-PCR. 
Results: SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2µM and 1.4µM, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21nM and 42nM, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6µM). 2-aminoethyldiphenyl borate (2APB) inhibited SOCE (IC(50) = 3.7µM) but enhanced basal influx (>100µM). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (=0.1µM). RT-PCR demonstrated transcript expression for ORAI 1, 2 and 3, and TRPC1, 3, 4 and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. 
Conclusion: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared to other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded. © 2012 John Wiley & Sons Ltd.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

Transient Ca2+-activated Cl-currents with endothelin in isolated arteriolar smooth muscle cells of the choroid

To characterize the effects of endothelin (ET)-1 on the Ca2+-activated Cl- conductance of choroidal arteriolar smooth muscle.

Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina

Rat retinae were dissociated to yield intact microvessels 7 to 42 microm in diameter. These were loaded with fura-2 AM and single fragments anchored down in a recording bath. Intracellular Ca(2+) levels from 20- to 30-microm sections of vessel were estimated by microfluorimetry. The vessels studied were identified as metarterioles and arterioles. Only the microvascular smooth muscle cells loaded with fura-2 AM and changes in the fluorescence signal were confined to these cells: Endothelial cells did not make any contribution to the fluorescence signal nor did they contribute to the actions of the drugs. Caffeine (10 mM) or elevated K(+) (100 mM) produced a transient rise in cell Ca(2+) in the larger vessels (diameters >18 microm) but had no effect on smaller vessels (diameters 30 min) on washing out the endothelin and the vessel failed to relax. These results demonstrate heterogeneity between smaller and larger retinal vessels with regard to Ca(2+) mobilisation and homogeneity with respect to the actions of vasoactive peptides.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells.

Experiments were performed to determine whether capacitative Ca(2+) entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca(2+)] was measured by imaging fura-2-loaded individual cells. Increases in the cytosolic [Ca(2+)] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine (RY)-sensitive SR Ca(2+) stores. In contrast, the cytosolic [Ca(2+)] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP(3) or RY pathways. The increase in the cytosolic [Ca(2+)] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca(2+)-free bathing solution. Rapid quenching of the fura-2 signal by 100 microM Mn(2+) following SR store depletion indicated that extracellular Ca(2+) entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP(3)- and RY-sensitive SR Ca(2+) stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP(3)- or RY-sensitive SR stores. Store depletion Ca(2+) entry in both pulmonary and renal ASMCs was strongly inhibited by Ni(2+) (0.1-10 mM), slightly inhibited by Cd(2+) (200-500 microM), but was not significantly affected by the voltage-gated Ca(2+) channel (VGCC) blocker nisoldipine (10 microM). The non-selective cation channel blocker Gd(3+) (100 microM) inhibited a portion of the Ca(2+) entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca(2+) store depletion activates CCE in parallel with the organization of intracellular Ca(2+) stores in canine pulmonary and renal ASMCs.

Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC50 of 6.76 nM.

Splicing of HDAC7 modulates the SRF-myocardin complex during stem-cell differentiation towards smooth muscle cells

Histone deacetylases (HDACs) have a central role in the regulation of gene expression. Here we investigated whether HDAC7 has an impact on embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs). ES cells were seeded on collagen-IV-coated flasks and cultured in the absence of leukemia inhibitory factor in differentiation medium to induce SMC differentiation. Western blots and double-immunofluorescence staining demonstrated that HDAC7 has a parallel expression pattern with SMC marker genes. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, overexpression of HDAC7 significantly increased beta-galactosidase-positive cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. We found that HDAC7 undergoes alternative splicing during ES cell differentiation. Platelet-derived growth factor enhanced ES cell differentiation into SMCs through upregulation of HDAC7 splicing. Further experiments revealed that HDAC7 splicing induced SMC differentiation through modulation of the SRF-myocardin complex. These findings suggest that HDAC7 splicing is important for SMC differentiation and vessel formation in embryonic development.

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

Splicing of histone deacetylase 7 modulates smooth muscle cell proliferation and neointima formation through nuclear β-catenin translocation

Vascular smooth muscle cell (SMC) proliferation has an indispensable role in the pathogenesis of vascular disease, but the mechanism is not fully elucidated. The epigenetic enzyme histone deacetylase 7 (HDAC7) is involved in endothelial homeostasis and SMC differentiation and could have a role in SMC proliferation. In this study, we sought to examine the effect of 2 HDAC7 isoforms on SMC proliferation and neointima formation.

Sp1-dependent activation of HDAC7 is required for platelet-derived growth factor-BB-induced smooth muscle cell differentiation from stem cells

We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.