Treatment with transducible phosphopeptide analogues of the small heat shock-related protein, HSP20, after experimental subarachnoid hemorrhage: prevention and reversal of delayed decreases in cerebral perfusion.

OBJECT: Delayed vasospasm is a significant cause of morbidity and mortality after subarachnoid hemorrhage (SAH). Proteomic therapeutics offers a new modality in which biologically active proteins or peptides are transduced into cells via covalent linkage to cell permeant peptides (CPPs). The hypothesis of this study was that either intrathecal or intravenous delivery of a phosphopeptide mimetic of the small heat shock-related protein, HSP20, linked to a CPP, would inhibit delayed decreases in cerebral perfusion after experimental SAH in a rat model. METHODS: This study was conducted in 3 parts: 1) prevention and 2) reversal of delayed decreases in cerebral perfusion via either intrathecal or intravenous administration of a CPP linked to phosphopeptide mimetics of HSP20 (AZX100) and 3) determining the effect of intravenous administration of AZX100 on blood pressure and heart rate. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Subsequently, AZX100 was administered intrathecally via a cisternal catheter or intravenously. Cerebral perfusion was determined by laser Doppler monitoring. Blood pressure was monitored by telemetry in a separate group of naïve animals treated with AZX100 for 24 hours. RESULTS: The maximal decrease in cerebral perfusion occurred 3 days after SAH. Cisternal administration of AZX100 (0.14-0.57 mg/kg) 24 hours after hemorrhage prevented decreases in cerebral perfusion after SAH. Animals receiving lower doses of AZX100 (0.068 mg/kg) or a scrambled sequence of the active HSP20 peptide linked to CPP developed decreases in cerebral perfusion similar to those seen in control animals. Intravenous administration of AZX100 (1.22 mg/kg) 24 hours after hemorrhage prevented the decreases in cerebral perfusion seen in the controls. Intravenous administration (0.175 mg/kg and 1.22 mg/kg) of AZX100 on Days 2 and 3 after SAH reversed decreases in cerebral perfusion as early as Day 3. There was no impact of AZX100 on blood pressure or heart rate at doses up to 2.73 mg/kg. CONCLUSIONS: Cisternal administration of AZX100 24 hours after hemorrhage prevented decreases in cerebral perfusion. Intravenous administration of AZX100 also prevented and reversed decreases in cerebral perfusion at doses that did not induce hypotension. Transduction of biologically active motifs of downstream regulators like HSP20 represents a potential novel treatment for SAH.

The small heat shock protein, HSPB6, in muscle function and disease.

The small heat shock protein, HSPB6, is a 17-kDa protein that belongs to the small heat shock protein family. HSPB6 was identified in the mid-1990s when it was recognized as a by-product of the purification of HSPB1 and HSPB5. HSPB6 is highly and constitutively expressed in smooth, cardiac, and skeletal muscle and plays a role in muscle function. This review will focus on the physiologic and biochemical properties of HSPB6 in smooth, cardiac, and skeletal muscle; the putative mechanisms of action; and therapeutic implications.

Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-beta1-induced CTGF expression in keloid fibroblasts.

A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation.

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

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.

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.

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.