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Biomedical subjects

Colleen M Brophy

Publications and source records attributed to Colleen M Brophy.

At least 19 recordsLinked to original sources
PubMed2006

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli.

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

3T3 Cells↗
PubMed2005

Short-term, moderate dosage Vitamin E supplementation may have no effect on platelet aggregation, coagulation profile, and bleeding time in healthy individuals.

OBJECTIVE: To investigate the in vivo effect of short-term, moderate dosage synthetic dl-alpha-tocopherol acetate supplementation on platelet aggregation, coagulation profile, and simulated bleeding time in healthy individuals. alpha-tocopherol is the most biologically active isomer of Vitamin E, traditionally promoted as an antioxidant and therapeutic agent in cardiovascular disease. In vitro studies have suggested that alpha-tocopherol plays a role in the inhibition of platelet aggregation. However, further investigations into the effect of alpha-tocopherol on bleeding in vivo have not duplicated these findings. MATERIALS AND METHODS: A total of 42 healthy volunteers complied with a 2-week abstinence period from the use of anti-platelet agents followed by determination of baseline platelet aggregation properties and coagulation studies using citrated whole blood. Moderate dosage Vitamin E (800 IU of dl-alpha-tocopherol acetate) was then self-administered for 14 days with reevaluation of platelet aggregation and coagulation profile, and simulated bleeding time after 14 days of Vitamin E supplementation. RESULTS: Forty subjects completed the 4-week study period. All 40 subjects demonstrated normal baseline coagulation studies and all had collagen-stimulated platelet aggregation assessment performed in triplicate. After Vitamin E supplementation, no significant difference was demonstrated in any study parameter. CONCLUSIONS: Dietary supplementation with moderate dosage synthetic dl-alpha-tocopherol acetate did not significantly prolong bleeding or platelet aggregation in vivo. The affect of Vitamin E on platelet aggregation in vitro does not appear to be reproducible in vivo. Therefore, peri-operative discontinuation of Vitamin E may not be necessary.

Adenosine Diphosphate↗
PubMed2005

Comparative study of the skin penetration of protein transduction domains and a conjugated peptide.

PURPOSE: We examined the ability of a protein transduction domain (PTD), YARA, to penetrate in the skin and carry a conjugated peptide, P20. The results with YARA were compared to those of a well-known PTD (TAT) and a control, nontransducing peptide (YKAc). The combined action of PTDs and lipid penetration enhancers was also tested. METHODS: YARA, TAT, YKAc, P20, YARA-P20, and TAT-P20 were synthesized by Fmoc chemistry. Porcine ear skin mounted in a Franz diffusion cell was used to assess the topical and transdermal delivery of fluorescently tagged peptides in the presence or absence of lipid penetration enhancers (monoolein or oleic acid). The peptide concentrations in the skin (topical delivery) and receptor phase (transdermal delivery) were assessed by spectrofluorimetry. Fluorescence microscopy was used to visualize the peptides in different skin layers. RESULTS: YARA and TAT, but not YKAc, penetrated abundantly in the skin and permeated modestly across this tissue. Monoolein and oleic acid did not enhance the topical and transdermal delivery of TAT or YARA but increased the topical delivery of YKAc. Importantly, YARA and TAT carried a conjugated peptide, P20, into the skin, but the transdermal delivery was very small. Fluorescence microscopy confirmed that free and conjugated PTDs reached viable layers of the skin. CONCLUSIONS: YARA and TAT penetrate in the porcine ear skin in vitro and carry a conjugated model peptide, P20, with them. Thus, the use of PTDs can be a useful strategy to increase topical delivery of peptides for treatment of cutaneous diseases.

Administration, Cutaneous↗
PubMed2005

Role of the small heat shock proteins in regulating vascular smooth muscle tone.

BACKGROUND: Vasospasm occurs in conduits used for vascular reconstructions. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle tone. Phosphorylated HSP20 is associated with vasorelaxation, and phosphorylated HSP27 inhibits the phosphorylation of HSP20 and relaxation. We hypothesized that the relationship between the phosphorylated states of these two proteins might dictate the tone of a vessel and may contribute to vasospasm. STUDY DESIGN: Sodium nitroprusside relaxation of vascular smooth muscle was recorded using pig coronary artery and human saphenous vein. Segments were frozen and homogenized, and extracted proteins were separated by one- and two-dimensional gel electrophoresis, transferred to Immobilon (Millipore), and probed with anti-cGMP-dependent protein kinase (anti-PKG), -HSP20, -HSP27, and -phosphoHSP27 antibodies. Band intensity was estimated using densitometry. RESULTS: Pig coronary artery completely relaxed (100%) with SNP (10(-7)M), but human saphenous vein only partially relaxed (20%). The levels of cGMP-dependent protein kinase and HSP20 were similar in the two tissue types. Human saphenous vein had significantly higher levels of HSP27 versus pig coronary artery (30.14 +/- 0.8 versus 6.62 +/- 0.2 pixels/mg; p < or = 0.001) and phosphoHSP27 (8.29 +/- 3.43 versus 0.012 +/- 0.008 pixels/mg; p < or = 0.001). CONCLUSIONS: Human saphenous vein contained significantly higher levels of HSP27 and pHSP27. Increased levels of phosphorylated HSP27 might contribute to vasospasm in human saphenous vein.

Animals↗
PubMed2005

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.

Amino Acid Sequence↗
PubMed2004

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

14-3-3 Proteins↗
PubMed2004

Sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of the heat shock-related protein 20 (HSP20).

PURPOSE: Sildenafil is an oral phosphodiesterase type 5 inhibitor that is a vasodilator used in the treatment of erectile dysfunction. Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of the heat shock related protein 20 (HSP20). The purpose of this study was to determine if sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20. MATERIALS AND METHODS: Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and sub-maximally contracted with serotonin. Increasing concentrations of sodium nitroprusside (SNP; 0.01-10 microM), sildenafil (0.01-100 microM), or PTD-pHSP20 (0.1-1.0 mM) were added to the baths and the percent relaxation was recorded. To determine if sildenafil-induced vasorelaxation was associated with increases in the phosphorylation of HSP20, rings of porcine coronary artery were untreated (control) or treated with SNP (10 microM) or sildenafil (100 microM) for 2, 5, and 10 min and then snap frozen. Extracted proteins were then separated using two-dimensional SDS-PAGE, transferred to a membrane, and probed for HSP20. RESULTS: Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose-dependent manner. Sildenafil-induced vasorelaxation was associated with an increase in the phosphorylation of HSP20. Transduction of peptide analogues of pHSP20 led to a dose-dependent relaxation of pre-contracted porcine coronary artery. CONCLUSIONS: These findings suggest that sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20 and that transduction of phosphopeptide analogues of HSP20 is sufficient for relaxation of vascular smooth muscle.

Animals↗
PubMed2004

Inactivity-induced modulation of Hsp20 and Hsp25 content in rat hindlimb muscles.

Denervation decreases small heat shock protein (HSP) content in the rat soleus muscle; however, it is unknown whether this change is due to inactivity or absence of a nerve-muscle connection. Spinal cord isolation (SI) is a model of inactivity with an intact neuromuscular connection. After 7 days of SI, Hsp20 and Hsp25 levels in the soleus, plantaris, and adductor longus muscles were lower than in control rats, whereas Hsp20 was unchanged and Hsp25 increased in the tibialis anterior. The results for the soleus indicate that these small HSPs respond to inactivity and that this response is not influenced by neural activity-independent factors. Furthermore, the data indicate that these HSPs are impacted to a greater degree in muscles that are predominantly slow or have an antigravity function than in flexor muscles. Understanding the regulation of these HSPs during chronic reductions in neuromuscular activity may have valuable applications for conditions such as spinal cord injury.

Animals↗
PubMed2004

Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstructions. However, vasospasm can lead to early graft failure. The leading cause of delayed graft failure is intimal hyperplasia. OBJECTIVE: To develop a proteomic approach to prevent vein-graft spasm and intimal hyperplasia. METHODS: Biomimetic peptide analogs of the small heat shock-related protein HSP20, containing a protein transduction domain (PTD), a phosphorylated serine, and a sequence of HSP20 surrounding the phosphorylation site (PTD-pHSP20), or a scrambled sequence of the same amino acids surrounding the phosphorylation site (PTD-scHSP20) were synthesized. The peptides were used in muscle bath and organ culture experiments with human saphenous vein (HSV) segments. Cultured smooth muscle cell lines were used to determine the effect of the peptides on proliferation and migration. RESULTS: In HSV rings precontracted with norepinephrine, PTD-pHSP20 but not PTD-scHSP20 led to relaxation. There was no significant difference in smooth muscle cell proliferation in cells treated with PTD-pHSP20 compared with PTD-scHSP20. Treatment with PTD-pHSP20 significantly inhibited cellular migration compared with PTD-scHSP20. Control, untreated, and PTD-scHSP20-treated saphenous veins had significant increases in intimal thickness after culture. This intimal thickening was completely inhibited by treatment with PTD-pHSP20. CONCLUSIONS: Protein transduction of biologically active motifs of HSP20 can affect pathologic and physiologic responses of HSV and represents a novel proteomic-based therapeutic approach. CLINICAL RELEVANCE: We have been a part of the genomics era and are now viewing the emergence of "proteomics." The genome is linear and relatively easy to examine; however the proteome is much more complex and dynamic. In essence, the purpose of gene therapy is to manipulate the genome to produce a particular protein. This manuscript describes a new proteomic approach in which the biologically active part of a protein is directly introduced into vascular cells. Peptides were synthesized which contained a total of 24 amino acids, 11 of which represent a protein transduction domain or "carrier" while the other 13 are the biologically active "cargo." These synthetic peptides prevent spasm (contraction) and intimal hyperplasia in segments of human saphenous vein treated ex vivo. Preclinical development is currently underway to develop these molecules as a proteomic-based vein harvest solution to enhance vein-graft patency.

Cells, Cultured↗
PubMed2004

Transducible recombinant small heat shock-related protein, HSP20, inhibits vasospasm and platelet aggregation.

BACKGROUND: Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstruction. Injury during harvest leads to vasospasm and a thrombogenic endoluminal surface. A proteomic transduction approach was developed to prevent vein graft vasospasm and thrombosis. METHODS: Recombinant HSP20 protein linked to the TAT protein transduction domain was generated in a bacterial expression system (TAT-HSP20). The effect of this protein on the inhibition of smooth muscle contraction was determined using rings of rabbit aorta and HSV in a muscle bath. In addition, the effects of TAT-HSP20 on platelet aggregation were determined in vitro using human citrated whole blood. RESULTS: Recombinant TAT-HSP20 inhibited norepinephrine-induced contraction of rabbit aortic and HSV segments. Similarly, TAT-HSP20 induced smooth muscle relaxation in HSV segments precontracted with norepinephrine. In human-citrated whole blood, platelet aggregation was significantly inhibited by TAT-HSP20 in a dose-dependent manner. CONCLUSIONS: The results of this study demonstrate that recombinant TAT-HSP20 inhibits vascular smooth muscle contraction and platelet aggregation. This suggests that HSP20 may be an ideal effector molecule to target as a proteomic approach to enhance early vein graft patency rates by preventing acute vasospasm and thrombosis.

Animals↗
PubMed2003

Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

Amino Acid Motifs↗
PubMed2003

The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin.

BACKGROUND: The heat shock-related protein (HSP) 20 is associated with actin and modulates smooth-muscle relaxation. We hypothesized that HSP20 mediates vasorelaxation via dynamic interactions with cytoskeletal proteins, such as actin, or actin binding proteins, such as alpha-actinin. METHODS: Physiological responses of strips of bovine carotid artery were analyzed with a muscle bath. In other experiments, the arteries were homogenized, and imunoprecipitations were performed. Immunohistochemistry with anti-HSP20 and anti-actinin antibodies was used to determine co-localization of the two proteins. RESULTS: Bovine carotid arteries contracted in response to serotonin and rapidly relaxed in response to forskolin. HSP20 co-immunoprecipitated with both actin and alpha-actinin, but not with HSP27 or paxillin. Immunostaining with HSP20 and alpha-actinin antibodies demonstrated that HSP20 and alpha-actinin co-localized. The amount of HSP20 that immunoprecipitated with alpha -actinin was markedly diminished in muscles that were treated with the vasorelaxant forskolin. CONCLUSIONS: HSP20 is associated with both actin and alpha-actinin. Activation of cyclic nucleotide-dependent signaling pathways leads to increases in the phosphorylation of HSP20 and a decrease in the association of HSP20 with alpha-actinin. These data suggest that phosphorylation of HSP20 may lead to relaxation of vascular smooth muscles through a dynamic association with cytoskeletal elements.

Actinin↗
PubMed2003

Causes, diagnosis, and management of duodenal obstruction after aortic surgery.

While ileus is common after repair of abdominal aortic aneurysm, small bowel obstruction is uncommon and duodenal obstruction is rare. We report a case of high-grade, mechanical obstruction of the third and fourth portions of the duodenum that required operative intervention. A review of the literature is included, with emphasis on the causes, diagnosis, and management options for duodenal obstruction associated with aortic reconstructive surgery.

Aged↗
PubMed2003

Phosphorylation of the heat shock-related protein, HSP20, mediates cyclic nucleotide-dependent relaxation.

Cyclic nucleotide-dependent relaxation of vascular smooth muscle is associated with increases in the phosphorylation of the small heat shock-related protein, HSP20. To determine whether phosphorylated HSP20 directly mediates relaxation, we used gene transfection and protein transduction of HSP20 analogues. Rat mesangial cells were transfected with constructs containing wild-type HSP20-enhanced green fluorescent protein (EGFP), phosphorylation site mutated HSP20 (S16A-HSP20-EGFP), or EGFP alone. Contractile properties were determined on a silicone polymer substrata. In the presence of serum, EGFP-vector transfected control cells and S16A-HSP20 transfected cells formed wrinkles on the polymer (contracted). Activation of cyclic nucleotide signaling pathways in the EGFP-vector transfected control cells led to a time-dependent decrease in the wrinkles (relaxation). The S16A-HSP20 transfected cells were refractory to cyclic nucleotide-dependent relaxation. Cells overexpressing the wild-type HSP20 did not form wrinkles on the polymer in response to serum (refractory to contraction). Treatment of precontracted strips of intact bovine carotid artery smooth muscle with synthetic peptides containing HIV-trans-activating transcriptional activator and a phosphopeptide motif of HSP20 led to dose-dependent relaxation. These data provide evidence that phosphorylated HSP20 has a direct role in smooth muscle relaxation and that small phosphopeptide motifs of HSP20 can mimic the effects of the entire molecule.

Animals↗
PubMed2002

Stress and vascular disease at the cellular and molecular levels.

The role of nitric oxide in mediating smooth muscle relaxation is well established. Less is known about the downstream signaling events inside the smooth muscle cell that produce relaxation of the contractile machinery. One possible effector molecule is the small heat shock protein HSP20. Increased phosphorylation of HSP20 is associated with vasorelaxation. HSP20 is associated with elements of the contractile machinery such as another small heat shock protein (HSP27), actin, and myosin. Thus HSP20 may mediate vasorelaxation by directly modulating cytoskeletal or contractile elements in muscle cells.

Animals↗