Nanomatrix Coated Stent Enhances Endothelialization but Reduces Platelet, Smooth Muscle Cell, and Monocyte Adhesion under Physiologic Conditions.

Cardiovascular disease is presently the number one cause of death worldwide. Current stents used to treat cardiovascular disease have a litany of unacceptable shortcomings: adverse clinical events including restenosis, neointimal hyperplasia, thrombosis, inflammation, and poor re-endothelialization. We have developed a biocompatible, multifunctional, peptide amphiphile-based nanomatrix coating for stents. In this study, we evaluated the ability of the nanomatrix coated stent to simultaneously address the issues facing current stents under physiological flow conditions in vitro. We found that the nanomatrix coated stent could increase endothelial cell migration, adhesion, and proliferation (potential for re-endothelialization), discourage smooth muscle cell migration and adhesion (potential to reduce neointimal hyperplasia and restenosis), and decrease both platelet activation and adhesion (potential to prevent thrombosis) as well as monocyte adhesion (potential to attenuate inflammatory responses) under physiological flow conditions in vitro. These promising results demonstrate the potential clinical utility of this nanomatrix stent coating, and highlight the importance of biocompatibility, multifunctionality, and bioactivity in cardiovascular device design.

Precision Medicine at the University of Alabama at Birmingham: Laying the Foundational Processes Through Implementation of Genotype-Guided Antiplatelet Therapy.

Precision medicine entails tailoring treatment based on patients' unique characteristics. As drug therapy constitutes the cornerstone of treatment for most chronic diseases, pharmacogenomics (PGx), the study of genetic variation influencing individual response to drugs, is an important component of precision medicine. Over the past decade investigations have identified genes and single-nucleotide polymorphisms (SNPs) and quantified their effect on drug response. Parallel development of point-of-care (POC) genotyping platforms has enabled the interrogation of the genes/SNPs within a timeline conducive to the provision of care. Despite these advances, the pace of integration of genotype-guided drug therapy (GGTx) into practice has faced significant challenges. These include difficulty in identifying SNPs with sufficiently robust evidence to guide clinical decision making, lack of clinician training on how to order and use genotype data, lack of clinical decision support (CDS) to guide treatment, and limited reimbursement. The University of Alabama at Birmingham's (UAB) efforts in precision medicine were initiated to address these challenges and improve the health of the racially diverse patients we treat.

Human postmortem device retrieval and analysis--orthopaedic, cardiovascular, and dental systems.

On the basis of decades of analyzing implant devices, tissues, and clinical records from revision surgical explants (called device failure), studies now include postmortem donors and in situ conditions (called success). A key issue has been information exchange from an interdisciplinary team where basic physical and biological studies complement details of the clinical conditions for each device. Overall, the summary information has shown that most revisions were based on factors associated with the patient health, disease, and compliance, with few outcomes directly correlated with technology and device-specific factors. However, because of the large numbers of devices implanted annually (millions), any sampling that reveals adverse circumstances could result in a high level of importance and the need for additional studies of this type. Experience from prior retrieval and analysis demonstrates significant value where peer reviewed results from investigations have altered the discipline and have improved the quality and longevity of health care associated with implanted devices. This report summarizes completed and ongoing studies of cardiovascular, dental, and orthopaedic systems. Endovascular stents from autopsies showed damage including fretting and corrosion from overlapping and intersecting conditions, plus some corrosion and element transfers to tissues from individual stents. Studies are proposed to increase numbers to evaluate clinical significance. Dental implants from postmortem donors that functioned more than 10 years provided evaluations of cobalt alloy devices and calcium phosphate bone graft substitutes originally investigated in the 1970s. Tissue integration and stability correlated with data from prior laboratory in vitro and in vivo investigations. Studies of articulation and fixation from orthopaedic total joint arthroplasties showed some limitations related to surface changes of YTZ zirconia, specific damage due to implantation procedures, which led to modified instrumentation and techniques, and several examples of conditions leading to longer-term device-to-bone fixation. These types of multidisciplinary studies are continuing.

Turbulent flow evaluation of the venous needle during hemodialysis.

Arteriovenous (AV) grafts and fistulas used for hemodialysis frequently develop intimal hyperplasia (IH) at the venous anastomosis of the graft, leading to flow-limiting stenosis, and ultimately to graft failure due to thrombosis. Although the high AV access blood flow has been implicated in the pathogenesis of graft stenosis, the potential role of needle turbulence during hemodialysis is relatively unexplored. High turbulent stresses from the needle jet that reach the venous anastomosis may contribute to endothelial denudation and vessel wall injury. This may trigger the molecular and cellular cascade involving platelet activation and IH, leading to eventual graft failure. In an in-vitro graft/needle model dye injection flow visualization was used for qualitative study of flow patterns, whereas laser Doppler velocimetry was used to compare the levels of turbulence at the venous anastomosis in the presence and absence of a venous needle jet. Considerably higher turbulence was observed downstream of the venous needle, in comparison to graft flow alone without the needle. While turbulent RMS remained around 0.1 m/s for the graft flow alone, turbulent RMS fluctuations downstream of the needle soared to 0.4-0.7 m/s at 2 cm from the tip of the needle and maintained values higher than 0.1 m/s up to 7-8 cm downstream. Turbulent intensities were 5-6 times greater in the presence of the needle, in comparison with graft flow alone. Since hemodialysis patients are exposed to needle turbulence for four hours three times a week, the role of post-venous needle turbulence may be important in the pathogenesis of AV graft complications. A better understanding of the role of needle turbulence in the mechanisms of AV graft failure may lead to improved design of AV grafts and venous needles associated with reduced turbulence, and to pharmacological interventions that attenuate IH and graft failure resulting from turbulence.

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.

Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus.

Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase, Nlk, that is a murine homolog of the Drosophila nemo (nmo) gene. The Nlk amino acid sequence is 54. 5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4. 0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60-70% of Nlk is localized to the nucleus, whereas 30-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.

Stent procedure complicated by thrombus formation distal to the lesion within a muscle bridge.

We report here a case of a patient who underwent percutaneous intervention to the left anterior descending artery, complicated by thrombus formation within the myocardial bridge distal to the lesion. There was complete angiographic resolution of thrombus and restoration of the normal antegrade blood flow after infusion of glycoprotein IIb/IIIa antagonist (abciximab). Our observation may suggest that the presence of myocardial bridging distal to coronary lesions should be considered seriously in preprocedural evaluation of the lesions as a potential risk factor for intracoronary thrombus formation. The main coronary arteries and the proximal segments of their major branches lie free on the epicardial surface of the heart. However, in some instances these vessels may penetrate into the muscle being surrounded by the myocardium, with the overlying muscle referred to as a "bridge". Myocardial bridging appears to be a congenital anomaly, due to failure of exteriorization of the primitive coronary intratrabecular arterial network. It occurs in 5-86% of patients in autopsy studies, and it is observed as systolic coronary artery narrowing in 0.5-12% of patients undergoing coronary arteriography. Although the gross anatomist had long recognized that the epicardial coronary artery might on occasion course directly through a segment of cardiac muscle, the physiological significance of this phenomenon was considered benign. This is partly because traditional teaching concerning coronary blood flow delivery to the left ventricular myocardium emphasized the primacy of the diastolic phase of the cardiac cycle. However, myocardial bridging is not always a benign finding, with recent reports suggesting an association with myocardial ischemia, infarction, vasospasm, cardiac arrythmias, and sudden death.

Differential expression of MEK1 and MEK2 during mouse development.

Map/Erk kinase 1 (MEK1) and MEK2 activate the Erk/ MAP kinases and have been implicated in cell growth and differentiation. To investigate the role of MEKs during mouse development, we have examined their expression and activity in various murine tissues during embryonic development and in the adult mouse. MEK2 RNA message is expressed at high levels in all embryonic tissues examined, including all neural tissues, and liver. This can be observed by in situ hybridization of tissue sections of 14.5-day-old mouse embryos, as well as by Northern blot analyses. MEK1, on the other hand, is expressed at very low levels in most embryonic murine tissue but can be detected in developing skeletal muscle. It is expressed at higher levels in adult tissue, particularly in brain, where it is expressed at high levels. Western blot analyses of MEK1 and MEK2 in 14.5-day-old embryonic and adult mouse tissue confirm the RNA analysis. Levels of MEK1 kinase activity are particularly high in adult brain tissues as well. These findings suggest that MEK2 may be the primary Erk/MAP kinase activator during development and that MEK1 may play a role in the proliferative or mitogenic response in adult mouse tissues. This study also raises the possibility that MEK1 and MEK2 might not have redundant functions in cells but may possess unique specificity in their interactions with upstream activators or downstream targets.

The 96 kDa protein kinase activated by oncogenic Ras in Xenopus egg extracts is also activated by constitutively active Mek: activation requires serine/threonine phosphorylation.

In the Xenopus egg and oocyte system, oncogenic Ras protein can induce cell cycle arrest. The effect of oncogenic Ras on the cell cycle seems to be mediated by the Raf-Mek-Erk pathway of Ras signal transduction since constitutively active Raf, Mek, or Erk can mimic the effect of oncogenic Ras protein and since specific inhibition of these kinases can block the effect of oncogenic Ras. Using activated Xenopus egg extracts, we previously reported that the cell cycle arrest induced by oncogenic Ras correlates with the activation of a 96 kDa protein that phosphorylates histone h2b in vitro. This result raised the possibility that the 96 kDa kinase (designated as p96h2bk) is a potential target of the Raf-Mek-Erk pathway that links the pathway to the control of the cell cycle. We report here that constitutively active Mek1 could activate p96h2bk in the absence of oncogenic Ras. Moreover, inhibition of endogenous Mek by a specific inhibitor, PD 098059, suppressed the activation of p96h2bk by oncogenic Ras. These results are consistent with the concept that p96h2bk is a component or target of the Raf-Mek-Erk pathway. Furthermore, we have shown that activation of p96h2bk requires serine/threonine phosphorylation of p96h2bk.

Intravascular ultrasound in the management of acute coronary artery dissection: a case report.

Crossing total occlusions is frequently difficult. The guidewire may enter a false lumen, thereby preventing successful balloon dilatations. We present a case of an acute arterial dissection following attempted angioplasty of a totally occluded right coronary artery. With an intravascular ultrasound probe in the false lumen, we were able to visualize a second guidewire and direct its passage into the true arterial lumen. This allowed for successful balloon dilatation and stent deployment restoring vessel patency.

MEK2 is a kinase related to MEK1 and is differentially expressed in murine tissues.

MEK1 is a dual specificity kinase that phosphorylates and activates the Erk/MAP kinases Erk-1 and Erk-2 by phosphorylating them on threonine and tyrosine. We report the cloning of a second MEK-like complementary DNA, Mek2, which predicts a protein of a molecular weight of 44,500. The MEK2 protein bears substantial sequence homology to MEK1, except at its amino terminus, and at a proline-rich region insert between the conserved kinase subdomains 9 and 10. MEK1 and MEK2 are shown to be encoded by different genes and are located on murine chromosomes 9 and 10, respectively. Northern analysis indicates that Mek2 is expressed at low levels in adult mouse brain and heart tissue, and at higher levels in other tissues examined. Low expression levels of Mek2 in brain tissue are in contrast to the high levels of Mek1 expressed in brain. Mek2 is expressed at high levels in neonatal brain, however. Recombinant MEK2 produced in bacteria phosphorylates a kinase-inactive Erk-1 on tyrosine and threonine, whereas a kinase-inactive mutant MEK2 does not. These findings suggest that MEK2 is a member of a multigene family.

Localization of the viral and cellular Src kinases to perinuclear vesicles in fibroblasts.

Previous studies have shown that the viral oncogene product, v-Src, is localized to a perinuclear structure. Here, we demonstrate by immunofluorescence analysis that the perinuclear structure corresponds to a local concentration of vesicles. Overexpressed normal cellular Src, c-Src, and a temperature-sensitive mutant of v-Src also are associated with these perinuclear vesicles. This perinuclear localization is observed in a variety of cell types using several different antibodies to Src, and it is independent of the fixation method. Immunoelectron ultracryomicroscopic analysis of rat fibroblast cells transformed by v-Src demonstrates an association of this protein with the limiting membranes of vesicles concentrated in the perinuclear region. These vesicles appear at the electron microscopic level to be multivesicular bodies on the basis of their size (0.3-1.0 microns in diameter), large electron-transparent lumens, and electron-dense vesicular inclusions. Morphometric analysis indicates that approximately 20% of the total cell v-Src protein is associated with these structures. This subpopulation of v-Src may have been recovered from the plasma membrane via the endocytotic pathway in a manner analogous to endocytosis of the epidermal growth factor receptor. Localization of the Src tyrosine kinases to these perinuclear endocytotic vesicles may be necessary for oncogenic transformation by v-Src and for normal functions of c-Src.

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.

Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.

GTPase-activating protein interactions with the viral and cellular Src kinases.

GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src.

Bioerodible polyanhydrides as drug-carrier matrices. I: Characterization, degradation, and release characteristics.

Polyanhydrides based on a variety of aromatic and aliphatic dicarboxylic acids were developed as bioerodible carrier matrices for controlled delivery applications. The high hydrolytic reactivity of the anhydride linkage provides an intrinsic advantage over other classes of bioerodible polymers in versatility and control of degradation rates. For example, using the poly[bis(p-carboxyphenoxy) alkane anhydrides] as models, polymers with degradation rates in the range of 10(-1) to 10(-4) mg/h/cm2 were obtained by changing the alkane from a methyl to a hexyl group. The polymers were characterized by infrared (IR), differential scanning calorimetry, gel permeation chromatography, and scanning electron microscopy (SEM). Near zero-order degradation kinetics were observed for the hydrophobic polyanhydrides over several months. The drug release profile of the model drug p-nitroaniline followed closely that of the degradation of injection-molded poly[bis(p-carboxyphenoxy) propane anhydride] over a period of more than 8 months. Close correlation of polymer degradation and drug release was also observed in other injection-molded samples (10% loading), suggesting a release mechanism that was dominantly degradation controlled. Degradation of these polyanhydrides was pH sensitive, being enhanced in high pH, and became more stable in acidic conditions.