Robert Lukowski

Transdifferentiation of Vascular Smooth Muscle Cells to Macrophage-Like Cells During Atherogenesis

Rationale: Atherosclerosis is a widespread and devastating disease, but the origins of cells within atherosclerotic plaques are not well defined. Objective: To investigate the specific contribution of vascular smooth muscle cells (SMCs) to atherosclerotic plaque formation by genetic inducible fate mapping in mice. Methods and Results: Vascular SMCs were genetically pulse-labeled using the tamoxifen-dependent Cre recombinase, CreERT2, expressed from the endogenous SM22&agr; locus combined with Cre-activatable reporter genes that were integrated into the ROSA26 locus. Mature SMCs in the arterial media were labeled by tamoxifen treatment of young apolipoprotein E–deficient mice before the development of atherosclerosis and then their fate was monitored in older atherosclerotic animals. We found that medial SMCs can undergo clonal expansion and convert to macrophage-like cells that have lost classic SMC marker expression and make up a major component of advanced atherosclerotic lesions. Conclusions: This study provides strong in vivo evidence for smooth muscle-to-macrophage transdifferentiation and supports an important role of SMC plasticity in atherogenesis. Targeting this type of SMC phenotypic conversion might be a novel strategy for the treatment of atherosclerosis, as well as other diseases with a smooth muscle component.

cGMP regulated protein kinases (cGK).

cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes--prkg1 and prkg2--code for cGKs, namely cGKI and cGKII. In mammals, two isozymes, cGKIalpha and cGKIbeta, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets, and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxta-glomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major downstream effectors of many, but not all signalling events of the NO/cGMP and the ANP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night, and endochondreal bone growth. cGKs are also modulators of cell growth and many other functions.

Rescue of cGMP Kinase I Knockout Mice by Smooth Muscle–Specific Expression of Either Isozyme

Smooth muscle expresses the I&agr; and the I&bgr; isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at ≈6 weeks. We reconstituted mice with the cGKI&agr; or -I&bgr; isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKI&agr; or the -I&bgr; coding sequences were placed under the control of the SM22&agr; promoter to express either isoform selectively in smooth muscle cells (SM-I&agr; or SM-I&bgr; transgene). To generate smooth muscle–specific cGKI&agr; or cGKI&bgr; rescue mice, the SM-I&agr; or SM-I&bgr; transgenes were crossed on a cGKI−/− genetic background. The levels of cGKI&agr; or -I&bgr; expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKI&agr; or -I&bgr; rescue mice, expression of the isozymes was not detectable in non–smooth muscle tissues and cells. Median survival time of the I&agr; and I&bgr; rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP–induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone- or K+-induced [Ca2+]i levels. The cGKI&agr; and cGKI&bgr; rescue mice did not show a significant difference in intestinal passage time of BaSO4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions.

Bile Acids Acutely Stimulate Insulin Secretion of Mouse β-Cells via Farnesoid X Receptor Activation and KATP Channel Inhibition

Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca2+ concentration ([Ca2+]c). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca2+]c and insulin secretion. FXR activation by TCDC is suggested to inhibit KATP current. The decline in KATP channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and KATP channel inhibition.

Cardiac hypertrophy is not amplified by deletion of cGMP-dependent protein kinase I in cardiomyocytes.

It has been suggested that cGMP kinase I (cGKI) dampens cardiac hypertrophy. We have compared the effect of isoproterenol (ISO) and transverse aortic constriction (TAC) on hypertrophy in WT [control (CTR)] mice, total cGKI-KO mice, and cGKIβ rescue mice (βRM) lacking cGKI specifically in cardiomyocytes (CMs). Infusion of ISO did not change the expression of cGKI in the hearts of CTR mice or βRM but raised the heart weight by ∼20% in both. An identical hypertrophic growth response was measured in CMs from CTR mice and βRM and in isolated adult CMs cultured with or without 1 μM ISO. In both genotypes, ISO infusion induced similar changes in the expression of hypertrophy-associated cardiac genes and significant elevation of serum atrial natriuretic peptide and total cardiac cGMP. No differences in cardiac hypertrophy were obtained by 7-day ISO infusion in 4- to 6-week-old conventional cGKI-KO and CTR mice. Furthermore, TAC-induced hypertrophy of CTR mice and βRM was not different and did not result in changes of the cGMP-hydrolyzing phosphodiesterase activities in hypertropic hearts or CMs. These results strongly suggest that cardiac myocyte cGKI does not affect the development of heart hypertrophy induced by pressure overload or chronic ISO infusion.

Ionizing radiation induces migration of glioblastoma cells by activating BK K+ channels

BACKGROUND AND PURPOSE Glioblastoma cells express high levels of Ca(2+)-activated BK K(+) channels which have been proposed to be indispensable for glioblastoma proliferation and migration. Since migration of glioblastoma cells is reportedly stimulated by ionizing radiation (IR), we tested for an IR-induced increase in BK channel activity and its effect on cell migration. MATERIALS AND METHODS T98G and U87MG cells were X-ray-irradiated with 0-2 Gy, BK channel activity was assessed by patch-clamp recording, migration by trans-well migration assay, and activation of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) by immunoblotting. RESULTS IR dose-dependently stimulated migration of glioblastoma cells which was sensitive to the BK channel inhibitor paxilline. Ca(2+)-permeabilization of T98G cells activated up to 350 BK channels per cells. Importantly, IR stimulated an increase in BK channel open probability but did not modify the total number of channels. Moreover, IR activated CaMKII in a paxilline-sensitive manner. Finally, inhibition of CaMKII by KN-93 abolished the IR-stimulated migration. CONCLUSIONS We conclude that IR stimulates BK channel activity which results in activation of CaMKII leading to enhanced glioblastoma cell migration.

KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK−/− cardiomyocytes. Transmission electron microscopy of BK−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

Background: Large-conductance potassium (BK) channels containing the stress axis regulated insert (STREX) are important regulators of cellular excitability. Results: Membrane detachment of STREX by protein kinase A (PKA)-mediated phosphorylation or de-palmitoylation established channel inhibition by protein kinase C (PKC). Conclusion: Membrane association of STREX regulates the access of PKC to its inhibitory phosphorylation site. Significance: The interplay of phosphorylation by PKA, PKC, and palmitoylation determines BK-STREX channel activity. Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ∼50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.

Role of Smooth Muscle cGMP/cGKI Signaling in Murine Vascular Restenosis

Background—Nitric oxide (NO) is of crucial importance for smooth muscle cell (SMC) function and exerts numerous, and sometimes opposing, effects on vascular restenosis. Although cGMP-dependent protein kinase type I (cGKI) is a principal effector of NO, the molecular pathway of vascular NO signaling in restenosis is unclear. The purpose of this study was to examine the functional role of the smooth muscle cGMP/cGKI signaling cascade in restenosis of vessels. Methods and Results—Tissue-specific mouse mutants were generated in which the cGKI protein was ablated in SMCs. We investigated whether the absence of cGKI in SMCs would affect vascular remodeling after carotid ligation or removal of the endothelium. No differences were detected between the tissue-specific cGKI mutants and control mice at different time points after vascular injury on a normolipidemic or apoE-deficient background. In line with these results, chronic drug treatment of injured control mice with the phosphodiesterase-5 inhibitor sildenafil elevated cGMP levels but had no influence on the ligation-induced remodeling. Conclusions—The genetic and pharmacological manipulation of the cGMP/cGKI signaling indicates that this pathway is not involved in the protective effects of NO, suggesting that NO affects vascular remodeling during restenosis via alternative mechanisms.

Roles of cGMP-dependent protein kinase I (cGKI) and PDE5 in the regulation of Ang II-induced cardiac hypertrophy and fibrosis

Significance It has been reported that elevation of cGMP and activation of cGMP-dependent protein kinase I (cGKI) by inhibition of PDE5 activity with sildenafil prevents cardiac hypertrophy. We studied the roles of cGKI and PDE5 inhibition on angiotensin (AII)-induced hypertrophy and fibrosis using control (Ctr) mice and mice expressing cGKI only in cells where the smooth muscle SM22α promotor is active (βRM). In Ctr mice, sildenafil did not reduce the AII-induced increase of cardiomyocyte (CM) size or myocyte hypertrophic markers but did reduce fibrosis. In βRM mice, sildenafil had little or no effect on markers of fibrosis. These results show that the sildenafil/cGMP/cGKI cascade can have an inhibitory effect on cardiac fibrosis but not on CM hypertrophy. Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIβ only in smooth muscle (βRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, βRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged βRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg⋅kg−1⋅d−1) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in βRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFβ, and CTGF mRNA in Ctr but not in βRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.