Transdifferentiation of human fibroblasts to endothelial cells: role of innate immunity.
2015; 131 (3): 300-309
Innate immunity and epigenetic plasticity in cellular reprogramming
CURRENT OPINION IN GENETICS & DEVELOPMENT
2014; 28: 89-91
-Cell fate is fluid, and may be altered experimentally by the forced expression of master regulators mediating cell lineage. Such reprogramming has been achieved using viral vectors encoding transcription factors. We recently discovered that the viral vectors are more than passive vehicles for transcription factors, as they participate actively in the process of nuclear reprogramming to pluripotency by increasing epigenetic plasticity. Based on this recognition, we hypothesized that small molecule activators of toll-like receptor 3 (TLR3), together with external microenvironmental cues that drive EC specification, might be sufficient to induce transdifferentiation of fibroblasts into ECs (iECs).-We show that TLR3 agonist Poly I:C, combined with exogenous EC growth factors, transdifferentiated human fibroblasts into ECs. These iECs were comparable to HMVEC in immunohistochemical, genetic and functional assays, including the ability to form capillary-like structures and to incorporate acetylated-LDL. Furthermore, iECs significantly improved limb perfusion and neovascularization in the murine ischemic hindlimb. Finally, using genetic knockdown studies, we find that the effective transdifferentiation of human fibroblasts to endothelial cells requires innate immune activation.-This study suggests that manipulation of innate immune signaling may be generally used to modify cell fate. As similar signaling pathways are activated by damage associated molecular patterns, epigenetic plasticity induced by innate immunity may play a fundamental role in transdifferentiation during wound healing and regeneration. Finally, this study is a first step toward development of a small molecule strategy for therapeutic transdifferentiation for vascular disease.
View details for DOI 10.1161/CIRCULATIONAHA.113.007394
View details for PubMedID 25359165
Therapeutic transdifferentiation: can we generate cardiac tissue rather than scar after myocardial injury?
Methodist DeBakey cardiovascular journal
2013; 9 (4): 210-212
Somatic cells can be reprogrammed to express the features of pluripotent cells, in that they can be differentiated into all three germ layers, and that they have the ability to replicate indefinitely. Recent studies suggest that the efficient induction of pluripotency requires the activation of innate immunity.
View details for DOI 10.1016/j.gde.2014.11.002
View details for Web of Science ID 000347764300015
View details for PubMedID 25461456
Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming
2012; 151 (3): 547-558
After myocardial injury, the cardiac muscle does not regenerate and heals by forming a scar. This process results in loss of heart function and ultimately heart failure. Recent application of reprogramming technology, where forced expression of master regulators convert scar-forming cells to become cardiovascular cells in vivo, has fueled new hope for the development of therapies targeting heart disease.
View details for PubMedID 24298312
Endothelial Cells Derived From Nuclear Reprogramming
2012; 111 (10): 1363-1375
Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripotent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved exceedingly inefficient. We discovered a striking difference in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogramming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain- and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables efficient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory pathways are required for efficient nuclear reprogramming in the induction of pluripotency.
View details for DOI 10.1016/j.cell.2012.09.034
View details for Web of Science ID 000310529300012
View details for PubMedID 23101625
Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance
2008; 103 (6): 606-614
The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health, the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell (EC) culture have been developed and refined. Recently, methods to derive ECs from pluripotent cells have extended the scientific range of human EC studies. Pluripotent stem cells may be generated, expanded, and then differentiated into ECs for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific ECs to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of ECs from stem cells; their characterization by genetic, histological, and functional studies; and their translational applications.
View details for DOI 10.1161/CIRCRESAHA.111.247213
View details for Web of Science ID 000310501300017
View details for PubMedID 23104878
Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (30): 12312-12317
Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.
View details for DOI 10.1161/CIRCRESAHA.108.175133
View details for Web of Science ID 000259252500011
View details for PubMedID 18669924
Induced pluripotent stem cells: how they will change the practice of cardiovascular medicine.
Methodist DeBakey cardiovascular journal
2013; 9 (4): 206-209
The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration- and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the alpha- and beta-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in alpha and C122 in beta, created mutants that were mostly resistant to desensitization. Structural analysis of the region near beta-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.
View details for DOI 10.1073/pnas.0703944104
View details for Web of Science ID 000248472100016
View details for PubMedID 17636120
Hypothalamic S-Nitrosylation Contributes to the Counter-Regulatory Response Impairment following Recurrent Hypoglycemia
2013; 8 (7)
Induced pluripotent stem cells (iPSCs) can be generated from adult somatic tissues by the forced expression of a few defined transcription factors, including Oct4, Sox2, Klf4, and c-Myc. iPSC technology holds tremendous promises for therapeutic cardiovascular regeneration because of the cells' unlimited capacity for proliferation and differentiation into all cell lineages. The iPSCs can be generated from somatic cells of patients with a genetic basis for their disease so as to understand the pathobiology of the disorder. This disease modeling can be adapted to high-throughput screens to discover new therapeutic molecules. Finally, the iPSC technology may enable personalized cell therapies, while avoiding the ethical concerns surrounding human embryonic stem cells. Intensive efforts are underway to develop reliable methods to guide stem cell differentiation into cardiovascular lineages in the treatment of peripheral artery disease and heart diseases. Studies of disease pathogenesis and drug discovery using iPSC technology shall advance the discovery of novel treatments for cardiovascular diseases.
View details for PubMedID 24298311
Leveraging the innate immunity pathway for transdifferentiation of fibroblasts to endothelial cells
2013; 18 (3): 153-154
NaHS relaxes rat cerebral artery in vitro via inhibition of L-type voltage-sensitive Ca2+ channel
2012; 65 (2): 239-246
Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.Acute insulin-hypoglycemia increased VMH ROS levels by 49±6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia.These data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.
View details for DOI 10.1371/journal.pone.0068709
View details for Web of Science ID 000322391400021
View details for PubMedID 23894333
Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2008; 28 (10): 1803-1810
H(2)S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K(+) channels. It is however unclear whether H(2)S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H(2)S donor inhibits voltage-sensitive Ca(2+) channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague-Dawley rats with the same potency against pre-contraction by 5-HT and 60 mmol/L KCl, which were unaffected by several K(+) channel blockers, N(G)-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl(2)-induced contraction in Ca(2+)-free, 60mM K(+)-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca(2+) currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca(2+)](i) in 60 mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H(2)S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine ?-lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca(2+) currents and suppressing [Ca(2+)](i) of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca(2+) influx via Ca(2+) channels.
View details for DOI 10.1016/j.phrs.2011.11.006
View details for Web of Science ID 000301868300012
View details for PubMedID 22133671
PAS-mediated dimerization of soluble guanylyl cyclase revealed by signal transduction histidine kinase domain crystal structure
JOURNAL OF BIOLOGICAL CHEMISTRY
2008; 283 (2): 1167-1178
Binding of nitric oxide (NO) to soluble guanylyl cyclase (sGC) leads to increased cGMP synthesis that activates cGMP-dependent protein kinase (PKG). Herein, we tested whether sGC activity is regulated by PKG.Overexpression of a constitutively active form of PKG (DeltaPKG) stimulated (32)P incorporation into the alpha1 subunit. Serine to alanine mutation of putative sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we observed that endogenous sGC phosphorylation on Ser 64 increases in cells and tissues exposed to NO, in a PKG-inhibitable manner. Wild-type (wt) sGC coexpressed with DeltaPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A alpha1/beta1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D alpha1 phosphomimetic /beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wild-type sGC. Expression of a phosphorylation-deficient sGC showed enhanced responsiveness to endothelium-derived NO, reduced desensitization to acute NO exposure, and allowed for greater VASP phosphorylation.We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.
View details for DOI 10.1161/ATVBAHA.108.165043
View details for Web of Science ID 000259278200020
View details for PubMedID 18635821
NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism
2007; 26 (2): 578-588
Signal transduction histidine kinases (STHK) are key for sensing environmental stresses, crucial for cell survival, and attain their sensing ability using small molecule binding domains. The N-terminal domain in an STHK from Nostoc punctiforme is of unknown function yet is homologous to the central region in soluble guanylyl cyclase (sGC), the main receptor for nitric oxide (NO). This domain is termed H-NOXA (or H-NOBA) because it is often associated with the heme-nitric oxide/oxygen binding (H-NOX) domain. A structure-function approach was taken to investigate the role of H-NOXA in STHK and sGC. We report the 2.1 A resolution crystal structure of the dimerized H-NOXA domain of STHK, which reveals a Per-Arnt-Sim (PAS) fold. The H-NOXA monomers dimerize in a parallel arrangement juxtaposing their N-terminal helices and preceding residues. Such PAS dimerization is similar to that previously observed for EcDOS, AvNifL, and RmFixL. Deletion of 7 N-terminal residues affected dimer organization. Alanine scanning mutagenesis in sGC indicates that the H-NOXA domains of sGC could adopt a similar dimer organization. Although most putative interface mutations did decrease sGCbeta1 H-NOXA homodimerization, heterodimerization of full-length heterodimeric sGC was mostly unaffected, likely due to the additional dimerization contacts of sGC in the coiled-coil and catalytic domains. Exceptions are mutations sGCalpha1 F285A and sGCbeta1 F217A, which each caused a drastic drop in NO stimulated activity, and mutations sGCalpha1 Q368A and sGCbeta1 Q309A, which resulted in both a complete lack of activity and heterodimerization. Our structural and mutational results provide new insights into sGC and STHK dimerization and overall architecture.
View details for DOI 10.1074/jbc.M706218200
View details for Web of Science ID 000252128100057
View details for PubMedID 18006497
Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.
View details for DOI 10.1038/sj.embol.7601521
View details for Web of Science ID 000243730700028
View details for PubMedID 17215864