Jeffrey P. Froehlich

Paclitaxel Stent Coating Inhibits Neointimal Hyperplasia at 4 Weeks in a Porcine Model of Coronary Restenosis

BackgroundDespite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. Methods and ResultsPalmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 &mgr;g/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P <0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P <0.05) at the highest level tested (187 &mgr;g/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control;P <0.05) with increasing dose (P <0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control;P <0.05) with escalating dose (P <0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. ConclusionsPaclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

Studies of sarcoplasmic reticulum function and contraction duration in young adult and aged rat myocardium

Abstract Kinetic measurements of Ca+2 uptake in microsomal fractions from rat myocardium demonstrated significantly lower rates of oxalate-facilitated accumulation in preparations from aged (24 to 25 month) hearts as compared to those from young adult (6 to 8 month) hearts. The per cent decline in transport activity in microsomes from aged hearts varied with Ca2+ concentration decreasing from 57% at 0.33 μ m Ca2+ to 24% at 1.21 μ m Ca2+. Double-reciprocal plots of the dependence of the velocity of accumulation on Ca2+ concentration showed upward curvature in both age groups indicating the presence of multiple Ca2+ binding sites. Mechanical studies using muscles isolated from the same hearts used to prepare sarcoplasmic reticulum demonstrated prolonged contraction duration in aged myocardium in agreement with previous findings. The lower in vitro rates of Ca2+ accumulation in aged microsomes suggest a possible biochemical mechanism to account for the observed increase in the time-course of cardiac relaxation.

Nitroxyl improves cellular heart function by directly enhancing cardiac sarcoplasmic reticulum Ca2+ cycling

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca2+. A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca2+ pump and the ryanodine receptor 2, leading to increased Ca2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca2+-release channels and accelerates Ca2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca2+ transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca2+ cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.

Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries

BACKGROUND Remodeling of the injured vascular wall is dependent on the action of several extracellular proteases. Previous studies have shown that expression of matrix metalloproteinases (MMP-2 and MMP-9) is upregulated after vascular injury and that MMP-2 is required for the migration of cultured vascular smooth muscle cells across complex extracellular matrix barriers. The present study examined changes in the expression of membrane-type metalloproteinase (MT-MMP-1), a putative regulator of MMP-2, in the tissue localization of MMP-2, and in the expression of activated and latent forms of MMP-2 and the tissue inhibitor of metalloproteinases, TIMP-2, in rat carotid arteries subjected to balloon catheter injury. METHODS AND RESULTS MT-MMP-1 mRNA levels increased sixfold after 3 days of injury, coinciding with an increase in MMP-2 activation assessed by gelatin zymography. Western blotting and gelatin zymography showed an increase in MMP-2 protein levels beginning 5 to 7 days after injury; immunocytochemistry and Western blotting showed that the increase occurred preferentially in the developing neointima. CONCLUSIONS These results show that increased expression of MT-MMP-1 and activation of MMP-2 occurs early after injury to the rat carotid artery and that at later times MMP-2 is preferentially localized to the developing neointima.

Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl.

Nitroxyl (HNO) donated by Angelis salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.

Diminished inotropic responsiveness to ouabain in aged rat myocardium.

We studied the effect of advanced age on the response to paired pacing and on the relationship between ouabain-induced inotropy and inhibition of (Na + K)-ATPas in hearts from young adult and senescent rats. In isometric trabeculae carneae, control values of developed tension (DT) and maximal rate of tension development (dT/dt) were not age-related. There was no age-related difference in the response to extrasystolic potentiation at prematurity intervals from 400 msec to the mechanical refractory period. The maximal response occurred at a prematurity interval of 200 msec and was above 200% of control. The inotropic response to ouabain occurred over a concentration range from 2 ∼ 10~6 to 6 ∼ 10′s M. DT and dT/dt in muscles from the young adult group exhibited a greater response than those from the senescent group; e.g., at 6 ∼ 10′s M both parameters were approximately four times greater in the former group. There was no age-dependent difference in ouabain-induced enzyme inhibition, which occurred over the same concentration range as did ouabain-induced alterations in mechanical function. These data indicate that paired pacing, but not ouabain, results in similar increases in inotropy and therefore similar increases in myoplasmic calcium concentration in young adult and aged rat myocardium. The age-dependent difference in ouabain responsiveness appears to be related to an age-associated alteration in a step other than enzyme inhibition linking ouabain binding to increased myoplasmic calcium levels. Circ Res 44: 517-523, 1979

A chemical quenching apparatus for studying rapid reactions

Abstract A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na + + K + )-stimulated ATPase.

Na + /H + exchanger: proton modifier site regulation of activity

The Na+/H+ exchangers (NHE1-6) are integral plasma membrane proteins that catalyze the exchange of extracellular Na+ for intracellular H+. In addition to Na+ and H+ transport sites, NHE has an intracellular allosteric H+ modifier site that increases exchange activity when occupied by H+. NHE activity is also subject to control by a variety of extrinsic factors including hormones, growth factors, cytokines, and pharmacological agents. Many of these factors, working through second messenger pathways acting directly or indirectly on NHE, regulate NHE activity by shifting the apparent affinity of the H+ modifier site to more alkaline or more acid pH. The underlying molecular mechanisms involved in the activation of NHE by the H+ modifier site are poorly understood at this time, but likely involve slow protein conformational changes within a NHE oligomer. In this paper, we present initial experiments measuring intracellular pH-dependent transition rates between active and inactive oligomeric conformations and describe how these transition rates may be important for overall regulation of NHE activity.

Intermolecular interactions in the mechanism of skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1): evidence for a triprotomer.

Native membrane sarcoplasmic reticulum (SR) Ca(2+)-ATPase isolated from skeletal muscle (SERCA1) exhibits oligomeric kinetic behavior [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. In the present study we used quenched-flow mixing, electron paramagnetic resonance (EPR), and chemical cross-linking to probe for intermolecular interactions at physiological (0.1 M) and high (0.4 M) KCl. Exposure of SR membranes to water- and lipid-soluble cross-linking reagents revealed a mixture of SERCA1 oligomeric species consisting mainly of dimers and trimers. Titration of iodoacetamide spin-labeled SERCA1 with AMPPCP in the presence of 10 microM Ca(2+) and 0.1 M KCl revealed high- (K(D) = 45 microM) and low-affinity (K(D) = 315 microM) nucleotide binding sites in a 2:1 ratio, respectively. Raising the [KCl] to 0.4 M increased the fraction of weak binding sites and lowered the K(D) of the high-affinity component (20 microM). Phosphorylation by 10 microM ATP at 21 degrees C and 0.1 M KCl produced an early burst of P(i) production without a corresponding decline in the steady-state phosphoenzyme (EP) level. The steady-state EP level was twice as large as the P(i) burst and received equal contributions from E1P and E2P. Chasing the phosphoenzyme at 0.4 M KCl and 2 degrees C with ADP revealed a biphasic time course of E1P formation with a slow phase that matched the kinetics of the transient EPR signal from the spin-labeled Ca(2+)-ATPase. The absence of a fast component in the EPR signal excludes E1P as its source. Instead, it arises from a slow, KCl-dependent transformation at the start of the cycle which controls the formation of downstream intermediates with an increased mole fraction of rotationally restricted probes. We modeled this behavior with a SERCA1 trimer in which the formation of E1P/E2/E2P from E1ATP/E2P/E1P results from concerted transformations in the subunits coupling phosphorylation (E1ATP --> E1P + ADP) to dephosphorylation (E2P --> E2 + P(i)) and the conversion of E1P to E2P.

Electrogenic and electroneutral partial reactions in Na+/K+ ATPase from eel electric organ

The generally accepted reaction scheme of the Na+/K+-ATPase is the Albers-Post cycle. It defines the reaction mechanism of the enzyme by a sequence of intermediates which are characterized by their chemical or structural properties, namely, conformation and state of phosphorylation. While much information has been accumulated about the reaction mechanism, much less is known about the transport mechanism of the Na+/K+-ATPase. An obvious strategy to address the latter problem is to correlate charge movement to the partial reactions in the Albers-Post scheme. The underlying assumption in this approach is that the charge movement reflects the movement of the transported Na+or K+ ions inside the protein.