Faculty Bibliography

Background: Current evidence suggests short-term right heart hemodynamic benefits of catheter directed thrombolysis (CDT) in patients treated with severe pulmonary embolism (PE). However, functional outcomes after CDT are lacking. We used 6-minute walk test (6MWT) as a marker of functional capacity after CDT and determined which factors were associated with 6MWT outcomes.
Method(s): This was a single-center retrospective study conducted from 2014-2018. Charts for patients who underwent 6MWT after CDT were reviewed. Three components of 6MWT, including distance walked, oxygen saturation during the test, and symptoms of dyspnea (Borg scale) were analyzed.
Result(s): 57 patients (mean age: 60 +/- 13 y, female/male: 27/57) underwent CDT; 56 patients had intermediate-risk PE, and 1 patient had high-risk PE. Total thrombolytic dose used was 19.66 +/- 8.27 mg. The right ventricular (RV) diameter decreased from 5.04 +/- 0.79 pre-CDT to 2.86 +/- 0.80 post-CDT echocardiogram (p < 0.001, n = 32), RV/left ventricular (LV) ratio decreased from 1.39 +/- 0.27 to 0.92 +/- 0.26 (p < 0.001, n = 32), pulmonary artery systolic pressure (PASP) decreased from 39.52 +/- 21.35 to 29.83 +/- 11.99 (p = 0.026, n = 23). 6MWT was performed 132.91 +/- 108.89 days after the procedure. The mean distance walked was 310 +/- 113.8 m. Multiple variables significantly correlated with 6MWT outcome, as shown in the Poster 15 Table. Multivariable regression analysis showed total thrombolytic dose (r² = 0.084, p = 0.032) and troponin (r² = 0.069, p = 0.04) as independent predictors of dyspnea scores before 6MWT.
Conclusion(s): Exercise capacity in patients with submassive PE treated with CDT is poor despite improvement in right heart hemodynamics. Total thrombolytic dose and troponin level are independent predictors for dyspnea scores before 6MWT

Background: Hospitalized patients often undergo interventional radiology (IR) procedures, many of which require individualized pre-procedure preparation and post-procedure care. Internists caring for these patients may not be familiar with requirements for these patients, causing procedural delays or periprocedural adverse events. Clear communication between IR and internal medicine is therefore necessary, but is often lacking.
Method(s): We conducted qualitative interviews with hospitalists, house staff, nurses and IR staff to identify common breakdowns in periprocedural communication between IR and referring medicine units. Utilizing insights from these interviews, we identified essential elements for pre-procedure and post-procedure communication. These elements were added as fields in templated pre-and post-procedure IR notes (Table 1). Each standardized template contains 16 elements. Outcome measures included proportion of key elements included in IR notes, inpatient provider satisfaction, and frequency of phone calls into and out of IR before and after the intervention.
Result(s): Before implementation of the standardized templates, pre-procedure consult notes (N=25) contained an average of 3.5 key elements (typically a brief medical history, assessment and plan), while post-implementation (N=50), these notes contained an average of 15.3 elements (p< 0.001). Similarly, post-procedure notes (N=25) contained an average of 4.7 elements (typically the names of the IR providers, a brief procedure description and patient condition) pre-intervention versus a mean of 15.0 elements post-intervention (N=50) (p< 0.001). Surveys of hospitalists pre-(N=17) and post-intervention (N=10) showed no significant difference in lack of confidence in preparing patients for IR procedures (52.9% vs. 30.0%, p=0.40), ineffective collaboration with IR (11.8% vs. 0%, p=0.44), and not receiving clear recommendations (35.3% vs. 10%, p=0.67); however analyses were underpowered. Total calls into and out of VIR decreased 15.6% overall (mean decrease of 7.7 calls/weekday and 24.5 calls/weekend, p=.006).
Conclusion(s): Standardizing pre-and post-procedure documentation can effectively increase the inclusion of key content, and this content may reduce internal medicine physician questions and concerns regarding periprocedural patient care

Our laboratory embarked on research to discover proteins the interaction of which with the mu opioid receptor (MOPr) is required for its function and regulation. We performed yeast two-hybrid screens, using the carboxy tail of the human MOPr as bait and a human brain library. This yielded a number of proteins that seemed to bind to the MOPr C-tail. The one we chose to study in detail was filamin A (FLNA). Evidence was obtained that there was indeed protein-protein binding between the C-tail of MOPr and FLNA. A human melanoma cell line (M2) lacking the gene for FLNA and a control cell line (A7) which differed from M2 only in having been transfected with the gene for FLNA and expressing the FLNA protein were made available to us. We transfected these cell lines with the gene for MOPr and used them in our studies. The absence of FLNA strongly reduced MOPr downregulation as well as desensitization of adenylyl cyclase inhibition and G protein activation. A recent finding, published here for the first time, is that FLNA is required for the activation by mu opioid agonists of the MAP kinase p38. Deletion studies indicated that the MOPr binding site on FLNA is in the 24th repeat, close to its C-terminal. It was further found that FLNA lacking the N-terminal actin binding domain is as capable as full length FLNA to restore cells to control status, suggesting that actin binding is not required. A surprising finding was that upregulation of MOPr by morphine and some agonist analogs occurs in M2 cells lacking FLNA, whereas normal receptor downregulation takes place in A7 cells

A number of dramatic breakthroughs in the neurobiology of addiction have occurred in the past 40 years. Two domains will be highlighted: the neurocircuitry of addiction and the molecular biology of addiction targets. The neurobiological substrates for the reinforcing effects of drugs of abuse have been largely identified both at the initial site of action and in the circuitry involved. In human imaging studies, decreases in dopaminergic function have been identified as a key element of addiction, lending support for research on the role of dopamine in addiction. Three novel areas currently are emerging: the role of deficits in frontal cortex functioning, changes in the brain neurocircuitry that convey long-term vulnerability to relapse, and the role of nondopaminergic systems in the neuroadaptations associated with the development of drug dependence. Parallel to these functional changes have been major advances in our understanding of the molecular biology of addiction; the greatest contribution has been in the understanding of the molecular mechanisms of opioid action. This paper reviews the major developments in our understanding of the molecular biology of the endogenous opioid system and the use of genomics to advance our knowledge of the function and regulation of opioid receptors and endorphins

We have previously reported that the protein filamin A (FLA) binds to the carboxyl tail of the mu opioid receptor (MOPr). Using human melanoma cells, which do not express filamin A, we showed that receptor down-regulation, functional desensitization and trafficking are deficient in the absence of FLA (Onoprishvili et al. Mol Pharmacol 64:1092-1100, 2003). Since FLA has a binding domain for actin and is a member of the family of actin cytoskeleton proteins, it is usually assumed that FLA functions via the actin cytoskeleton. We decided to test this hypothesis by preparing cDNA coding for mutant FLA lacking the actin binding domain (FLA-ABD) and expressing FLA-ABD in the human melanoma cell line M2 (M2-ABD cell line). We report here that this mutant is capable of restoring almost as well as full length FLA the down-regulation of the human MOPr. It is similarly very effective in restoring functional desensitization of MOPr, as assessed by the decrease in G-protein activation after chronic exposure of M2-ABD cells to the mu agonist DAMGO. We also found that A7 cells, expressing wild type FLA, exhibit rapid activation of the MAP kinases, ERK 1 and 2, by DAMGO, as shown by a rise in the level of phospho-ERK 1 and 2. This is followed by rapid dephosphorylation (inactivation), which reaches basal level between 30 and 60 min after DAMGO treatment. M2 cells show normal activation of ERK 1 and 2 in the presence of DAMGO, but very slow inactivation. The rapid rate of MAPK inactivation is partially restored by FLA-ABD. We conclude that some functions of FLA do not act via the actin cytoskeleton. It is likely that other functions, not studied here, may require functional binding of the MOPr-FLA complex to actin

We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a subclone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed that after chronic morphine treatment (24 h) of A7 cells, MOP-binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188% of control naive cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced up-regulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e., it decreased DAMGO-induced stimulation of GTPgammaS binding. In M2 cells DAMGO stimulation of GTPgammaS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture

A yeast two-hybrid screen, using the carboxyl tail of the human mu opioid receptor as bait and a human brain cDNA library as target, indicated that the carboxyl terminal portion of hlj1, a member of the human heat shock protein 40 family, interacts with the carboxyl tail of the human mu opioid receptor. To determine if direct in vitro binding occurs between these two proteins, we performed overlay experiments. Results from the overlay experiments showed that binding occurs between the His fusion protein of hlj1 and the GST fusion protein of the carboxyl tail of the human mu opioid receptor. In contrast, no binding with the His fusion protein of hlj1 occurred with GST alone or the GST fusion protein of the third cytoplasmic loop of the human mu opioid receptor. Results from co-immunoprecipitation studies, carried out in whole HEK cell lysates, confirmed in vivo binding between these two proteins. Immunofluorescent studies, using laser scanning confocal microscopy, showed significant co-localization between hlj1 and the human mu opioid receptor in the cell membrane. The function of this protein-protein interaction and its physiological relevance in animal and human brain is yet to be determined

The carboxyl tail of the human mu opioid receptor was shown to bind the carboxyl terminal region of human filamin A, a protein known to couple membrane proteins to actin. Results from yeast two-hybrid screening were confirmed by direct protein-protein binding and by coimmunoprecipitation of filamin and mu opioid receptor from cell lysates. To investigate the role of filamin A in opioid receptor function and regulation, we used the melanoma cell line M2, which does not express filamin A, and its subclone A7, transfected with human filamin A cDNA. Both cell lines were stably transfected with cDNA encoding myc-tagged human mu opioid receptor. Fluorescent studies, using confocal microscopy, provided evidence that filamin and mu opioid receptors were extensively colocalized on the membranes of filamin-expressing melanoma cells. The immunostaining of mu opioid receptors indicated that the lack of filamin had no detectable effect on membrane localization of the receptors. Moreover, mu opioid receptors function normally in the absence of filamin A, as evidenced by studies of opioid binding and DAMGO inhibition of forskolin-stimulated adenylyl cyclase. However, agonist-induced receptor down-regulation and functional desensitization were virtually abolished in cells lacking filamin A. The level of internalized mu-opioid receptors, after 30-min exposure to agonist, was greatly reduced, suggesting a role for filamin in mu opioid receptor trafficking. During these studies, we observed that forskolin activation of adenylyl cyclase was greatly reduced in filamin-lacking cells. An even more unexpected finding was the ability of long-term treatment with [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin of M2 cells, containing mu opioid receptors, to restore normal forskolin activation. The mechanism of this effect is currently unknown. It is postulated that the observed effects on mu opioid receptor regulation by filamin A and, by implication, of the actin cytoskeleton may be the result of its role in mu opioid receptor trafficking