Rüdiger Popp

Transdifferentiation of Blood-Derived Human Adult Endothelial Progenitor Cells Into Functionally Active Cardiomyocytes

Background—Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent studies suggest that progenitor cells can transdifferentiate into other lineages. However, the transdifferentiation potential of endothelial progenitor cells (EPCs) is unknown. Methods and Results—EPCs were obtained from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Diacetylated LDL–labeled EPCs were then cocultivated with rat cardiomyocytes for 6 days, resulting in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94±1.39% and 5.04±1.09% of EPCs from healthy adults (n=15) or CAD patients (n=14, P <0.01 versus healthy adults), respectively, expressed &agr;-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed &agr;-sarcomeric actinin, cardiac troponin I (both with partial sarcomeric organization), atrial natriuretic peptide, and myocyte enhancer factor 2. Fluo 4 imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in transdifferentiated human EPCs. Single-cell microinjection of Lucifer yellow and calcein-AM labeling of cardiomyocytes demonstrated gap junctional communication between 51±7% of EPCs (16 hours after labeling, n=4) and cardiomyocytes. EPC transdifferentiation into cardiomyocytes was not observed with conditioned medium but in coculture with paraformaldehyde-fixed cardiomyocytes. Conclusions—EPCs from healthy volunteers and CAD patients can transdifferentiate in vitro into functionally active cardiomyocytes when cocultivated with rat cardiomyocytes. Cell-to-cell contact but not cellular fusion mediates EPC transdifferentiation. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

A transferable, beta‐naphthoflavone‐inducible, hyperpolarizing factor is synthesized by native and cultured porcine coronary endothelial cells.

1. The vascular endothelium releases a hyperpolarizing factor (endothelium‐derived hyperpolarizing factor, EDHF) tentatively identified as a cytochrome P450‐derived arachidonic acid metabolite. However, there is still controversy concerning its transferability and identity. We designed a bioassay system for assessing EDHF release in which the membrane potential was recorded in cultured vascular smooth muscle cells located downstream from donor endothelial cells. 2. Under combined nitric oxide (NO) synthase and cyclo‐oxygenase blockade with NG‐nitro‐L‐arginine (100 mumol l‐1) and diclofenac (10 mumol l‐1), the superfusate from bradykinin (30 mumol l‐1)‐stimulated, cultured porcine coronary endothelial cells induced a distinct hyperpolarization followed by a depolarization. Direct application of bradykinin to the smooth muscle cells resulted solely in membrane depolarization. Similar results were obtained using bradykinin‐stimulated porcine coronary arteries as donor. 3. Single‐channel current measurements suggest that this EDHF‐induced hyperpolarization was elicited by the activation of Ca(2+)‐dependent K+ channels. 4. Increasing the transmural pressure within the donor segment significantly enhanced the duration, but not the amplitude of the hyperpolarization induced by the effluate from bradykinin‐stimulated donor segments. 5. Inhibition of P450 oxygenase activity with clotrimazole (3 mumol l‐1) or 17‐octadecynoic acid (3 mumol l‐1) abolished EDHF release from the coronary endothelium, while the P450‐derived arachidonic acid metabolite, 5,6‐epoxyeicosatrienoic acid, induced a hyperpolarization of detector smooth muscle cells almost identical to that induced by EDHF. Moreover, induction of P450 activity by beta‐naphthoflavone (3 mumol l‐1, 48 h), significantly increased the bradykinin‐induced release of EDHF. 6. These findings suggest that the vascular endothelium releases a transferable hyperpolarizing factor, chemically distinct from NO and prostacyclin, in response to agonists and mechanical stimulation. This beta‐naphthoflavone‐inducible EDHF appears to be a cytochrome P450‐derived metabolite of arachidonic acid, which elicits hyperpolarization by activation of Ca(2+)‐dependent K+ channels.

Epoxyeicosatrienoic Acids Regulate Trp Channel–Dependent Ca2+ Signaling and Hyperpolarization in Endothelial Cells

Objective—An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells. Methods and Results—In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca2+ influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition. Similar effects were observed in cultured endothelial cells overexpressing CYP2C9, but not in CYP2C9-deficient cells, and were prevented by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid as well as by the cAMP antagonist, Rp-cAMPS. The effects on Ca2+ were mirrored by prolongation of the bradykinin-induced hyperpolarization. Ruthenium red and the combination of charybdotoxin and apamin prevented the latter effect, suggesting that Trp channel activation increases Ca2+ influx and prolongs the activation of Ca2+-dependent K+ (KCa) channels. Indeed, overexpression of CYP2C9 enhanced the agonist-induced translocation of a TrpC6-V5 fusion protein to caveolin-1–rich areas of the endothelial cell membrane, which was prevented by Rp-cAMPS and mimicked by 11,12-EET. Conclusions—Elevated EET levels regulate Ca2+ influx into endothelial cells and the subsequent activation of KCa channels, via a cAMP/PKA-dependent mechanism that involves the intracellular translocation of Trp channels.

Nifedipine Increases Cytochrome P4502C Expression and Endothelium-Derived Hyperpolarizing Factor–Mediated Responses in Coronary Arteries

In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.

Pulsatile Stretch in Coronary Arteries Elicits Release of Endothelium-Derived Hyperpolarizing Factor: A Modulator of Arterial Compliance

To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca2+-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N(G)-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid

Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor–sensitive and NO- and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C–expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-&bgr;-d-ribofuranosylbenzimidazole 3′,5′-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal–regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2.

Proinflammatory Mediators Chronically Downregulate the Formation of the Endothelium-Derived Hyperpolarizing Factor in Arteries Via a Nitric Oxide/Cyclic GMP–Dependent Mechanism

BACKGROUND Endothelium-dependent dilator responses mediated by NO and endothelium-derived hyperpolarizing factor (EDHF) are altered in arteriosclerosis and sepsis. The possibility that proinflammatory mediators that stimulate the expression of inducible NO synthase (NOS II) affect the generation of EDHF was examined in isolated arteries. METHODS AND RESULTS Under combined blockade of NOS and cyclooxygenase, EDHF-mediated relaxation elicited by several agonists was significantly attenuated in rabbit carotid and porcine coronary arteries exposed to cytokines and lipopolysaccharide. The blunted relaxation was coincident with NOS II expression and was prevented by inhibition of NOS II as well as of global protein synthesis. The NO donor CAS 1609 and 8-bromo-cGMP mimicked the proinflammatory mediator effect. In contrast, long-term blockade of endothelial NO generation increased the relaxation in carotid but not in coronary arteries. Proinflammatory mediators reduced the synthesis of EDHF assessed as hyperpolarization of vascular smooth muscle cells elicited by the effluent from bradykinin-stimulated coronary arteries. Proinflammatory mediators induced NOS II expression in cultured endothelial cells and decreased the expression of cytochrome P450 enzymes, which are the most probable candidates for the synthesis of EDHF. CONCLUSIONS Proinflammatory mediators inhibit the formation of EDHF in isolated arteries. This impairment is coincident with NOS II expression in the arterial wall and seems to be mediated through the induced generation of NO, which downregulates the putative EDHF-forming enzyme. Thus, a decreased formation of EDHF may contribute to the endothelial dysfunction in arteriosclerosis and sepsis.

Hyperthyroidism enhances endothelium-dependent relaxation in the rat renal artery

OBJECTIVE Hyperthyroidism has pronounced effects on vascular function and endothelium-dependent relaxation. The aim of the present study was to identify mechanisms underlying hyperthyroidism-induced alterations in endothelial function in rats. METHODS Animals were subjected to either a single injection (36 h) or 8 weeks treatment with the thyroid hormone triiodothyronine (T3, i.p.). Vascular reactivity and agonist-induced hyperpolarization were studied in isolated renal arteries. Endothelial nitric oxide (NO) synthase expression and cyclic AMP accumulation were determined in aortic segments. RESULTS Endothelium-dependent relaxations to acetylcholine (ACh) were enhanced by T3 36 h after injection and after treatment for 8 weeks. Thirty-six hours after T3 application, relaxation mediated by the endothelium-derived hyperpolarizing factor (EDHF) and by endothelium-derived NO were significantly enhanced. After 8 weeks treatment with T3, however, EDHF-mediated relaxation was impaired, whereas NO-mediated relaxation remained enhanced. KCl- and ACh-induced hyperpolarizations were more pronounced in arteries from rats treated with T3 for 36 h compared to control, whereas in arteries from rats treated with T3 for 8 weeks both responses were attenuated. In rats treated for 36 h, vascular cyclic AMP levels were enhanced in the aorta and inhibition of protein kinase A attenuated EDHF-mediated relaxations of the renal artery without affecting responses in arteries from the control group. In the aorta from rats treated with T3 for 8 weeks, the expression of the endothelial NO synthase was markedly up-regulated (463+/-68%). CONCLUSIONS These data indicate that short-term treatment with T3 increases endothelium-dependent relaxation, most probably by increasing vascular cyclic AMP content. Following treatment with T3 for 8 weeks, expression of the endothelial NO synthase was enhanced. During this phase, NO appears to be the predominant endothelium-derived vasodilator.

The extracellular regulated kinases (ERK) 1/2 mediate cannabinoid-induced inhibition of gap junctional communication in endothelial cells.

Cannabinoids are potent inhibitors of endothelium‐derived hyperpolarizing factor (EDHF)‐mediated relaxations. We set out to study the mechanism underlying this effect and the possible role of cannabinoid‐induced changes in intercellular gap junction communication. In cultured endothelial cells, Δ9‐tetrahydrocannabinol (Δ9‐THC) and the cannabinoid receptor agonist HU210, increased the phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) and inhibited gap junctional communication, as determined by Lucifer Yellow dye transfer and electrical capacity measurements. Δ9‐THC elicited a pronounced increase in the phosphorylation of connexin 43, which was sensitive to PD98059 and U0126, two inhibitors of ERK1/2 activation. Inhibition of ERK1/2 also prevented the Δ9‐THC‐induced inhibition of gap junctional communication. Δ9‐THC prevented both the bradykinin‐induced hyperpolarization and the nitric oxide and prostacyclin‐independent relaxation of pre‐contracted rings of porcine coronary artery. These effects were prevented by PD98059 as well as U0126. In the absence of Δ9‐THC, neither PD98059 nor U0126 affected the NO‐mediated relaxation of coronary artery rings but both substances induced a leftward shift in the concentration – relaxation curve to bradykinin when diclofenac and Nωnitro‐L‐arginine were present. Moreover, PD98059 and U0126 prolonged the bradykinin‐induced hyperpolarization of porcine coronary arteries, without affecting the magnitude of the response. These results indicate that the cannabinoid‐induced activation of ERK1/2, which leads to the phosphorylation of connexin 43 and inhibition of gap junctional communication, may partially account for the Δ9‐THC‐induced inhibition of EDHF‐mediated relaxation. Moreover, the activation of ERK1/2 by endothelial cell agonists such as bradykinin, appears to exert a negative feedback inhibition on EDHF‐mediated responses.

Caffeine Enhances Endothelial Repair by an AMPK-Dependent Mechanism

Objective—Migratory capacity of endothelial progenitor cells (EPCs) and mature endothelial cells (ECs) is a key prerequisite for endothelial repair after denuding injury or endothelial damage. Methods and Results—We demonstrate that caffeine in physiologically relevant concentrations (50 to 100 &mgr;mol/L) induces migration of human EPCs as well as mature ECs. In patients with coronary artery disease (CAD), caffeinated coffee increased caffeine serum concentration from 2 &mgr;mol/L to 23 &mgr;mol/L, coinciding with a significant increase in migratory activity of patient-derived EPCs. Decaffeinated coffee neither affected caffeine serum levels nor migratory capacity of EPCs. Treatment with caffeine for 7 to 10 days in a mouse-model improved endothelial repair after denudation of the carotid artery. The enhancement of reendothelialization by caffeine was significantly reduced in AMPK knockout mice compared to wild-type animals. Transplantation of wild-type and AMPK−/− bone marrow into wild-type mice revealed no difference in caffeine challenged reendothelialization. ECs which were depleted of mitochondrial DNA did not migrate when challenged with caffeine, suggesting a potential role for mitochondria in caffeine-dependent migration. Conclusion—These results provide evidence that caffeine enhances endothelial cell migration and reendothelialization in part through an AMPK-dependent mechanism, suggesting a beneficial role for caffeine in endothelial repair.