作者: Weisbart, Richard H ; Jordaan, Gwen ; Saunders, Jacquelyn T ; Gera, Joseph ; Nishimura, Robert N ; Chang, Sophia S ; Chan, Grace ; Hansen, James E ; Hino, Chris ; Bashir, Mohammad T

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H₂O₂. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 μM concentrations after two different concentrations of H₂O₂ treatment. All three recombinant proteins protected SH-SY5Y cells from acute H₂O₂ toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H₂O₂ resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

2017-09-01·Journal of the American College of Cardiology1区 · 医学

Cardioprotective Effects of HSP72 Administration on Ischemia-Reperfusion Injury

1区 · 医学

Article

作者: Francis G. Blankenberg ; H. William Strauss ; Takashi Tanimoto ; Takehiro Nakahara ; Nezam Haider ; Hideki Kawai ; Dongbin Kim ; Partho P. Sengupta ; Grace Chan ; Richard A. Richieri ; Takashi Akasaka ; Glenn T. Reynolds ; Navneet Narula ; John Billimek ; Missag H. Parseghian ; Artiom D. Petrov ; Richard H. Weisbart ; Jagat Narula ; Farhan Chaudhry ; Robert Nishimura

BACKGROUND:

Although early reperfusion is the most desirable intervention after ischemic myocardial insult, it may add to damage through oxidative stress.

OBJECTIVES:

This study investigated the cardioprotective effects of a single intravenous dose of heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv) in a rabbit ischemia-reperfusion model. The Fv facilitates rapid transport of HSP72 into cells, even with intact membranes.

METHODS:

A left coronary artery occlusion (40 min) reperfusion (3 h) model was used in 31 rabbits. Of these, 12 rabbits received the fusion protein (Fv-HSP72) intravenously. The remaining 19 control rabbits received a molar equivalent of 3E10Fv alone (n = 6), HSP72 alone (n = 6), or phosphate-buffered saline (n = 7). Serial echocardiographic examinations were performed to assess left ventricular function before and after reperfusion. Micro-single-photon emission computed tomography imaging of ^99mTc-labeled annexin-V was performed with micro-computed tomography scanning to characterize apoptotic damage in vivo, followed by gamma counting of the excised myocardial specimens to quantify cell death. Histopathological characterization of the myocardial tissue and sequential cardiac troponin I measurements were also undertaken.

RESULTS:

Myocardial annexin-V uptake was 43% lower in the area at risk (p = 0.0003) in Fv-HSP72-treated rabbits compared with control animals receiving HSP72 or 3E10Fv alone. During reperfusion, troponin I release was 42% lower and the echocardiographic left ventricular ejection fraction 27% higher in the Fv-HSP72-treated group compared with control animals. Histopathological analyses confirmed penetration of 3E10Fv-containing molecules into cardiomyocytes in vivo, and treatment with Fv-HSP72 showed fewer apoptotic nuclei compared with control rabbits.

CONCLUSIONS:

Single-dose administration of Fv-HSP72 fusion protein at the time of reperfusion reduced myocardial apoptosis by almost one-half and improved left ventricular functional recovery after myocardial ischemia-reperfusion injury in rabbits. It might have potential to serve as an adjunct to early reperfusion in the management of myocardial infarction.

2010-03-01·Stroke1区 · 医学

Recombinant Fv-Hsp70 Protein Mediates Neuroprotection After Focal Cerebral Ischemia in Rats

1区 · 医学

Article

作者: Zhan, Xinhua ; Kim, Chester ; Liao, Isaac H. ; Hansen, James E. ; Sharp, Frank R. ; Nishimura, Robert N. ; Weisbart, Richard H. ; Ander, Bradley P. ; Clements, Douglas

Background and Purpose— This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior after experimental ischemic stroke. Methods— Focal cerebral ischemia was produced by occluding the middle cerebral artery using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 hours. At 2¼ hours and 3 hours after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg/kg) or saline was injected through the tail vein. Sensorimotor function and infarction volume were assessed at 24 hours after ischemia. Results— Administration of Fv-Hsp70 after focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensorimotor function compared with the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. Conclusion— Fv-Hsp70 protected the ischemic brain in this experimental stroke model.

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