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Study of Protein Expression in Peri-Infarct Tissue after Cerebral Ischemia.

Brea D, Agulla J, Staes A, Gevaert K, Campos F, Sobrino T, Blanco M, D√°valos A, Castillo J, Ramos-Cabrer P - Sci Rep (2015)

Bottom Line: Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue.Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain.Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain.

View Article: PubMed Central - PubMed

Affiliation: 1] Neurology Department, Neurovascular Area, Clinical Neurosciences Research Laboratory, University Clinical Hospital, Health Research Institute of Santiago de Compostela (IDIS), University of Santiago de Compostela, Spain [2] Cellular and Molecular Neurobiology Research Group and Grup de Recerça en Neurociencies del IGTP, Department of Neurosciences, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias I Pujol-Universitat Autónoma de Barcelona, Badalona, Spain.

ABSTRACT
In this work, we report our study of protein expression in rat peri-infarct tissue, 48 h after the induction of permanent focal cerebral ischemia. Two proteomic approaches, gel electrophoresis with mass spectrometry and combined fractional diagonal chromatography (COFRADIC), were performed using tissue samples from the periphery of the induced cerebral ischemic lesions, using tissue from the contra-lateral hemisphere as a control. Several protein spots (3408) were identified by gel electrophoresis, and 11 showed significant differences in expression between peri-infarct and contra-lateral tissues (at least 3-fold, p < 0.05). Using COFRADIC, 5412 proteins were identified, with 72 showing a difference in expression. Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue. Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain. Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain.

No MeSH data available.


Related in: MedlinePlus

Western blot (WB) analysis of the expression of the 70‚ÄČkDa Heat Shock Protein (HSP70) in the ischemic brain.Top) Cropped band of the WB gel corresponding to HSP70 in control tissue (CL, contra-lateral hemisphere), peri-infarct (PI), and the core of the ischemic lesion (L) from the 1D western blot (isoelectric point). All 3 samples were run together in the same gel. ő≤-Actin was used as the loading control (cropped band shown) in the same gel and at the same time (complete gels presented in supplementary data 1). Middle) Quantification of the expression levels, presented as the HSP70/Actin ratio (mean‚ÄȬĪ‚ÄČSDEV of pooled data from two different rats). Bottom) Cropped band corresponding to the second dimension of the western blot study over 2D electrophoresis gels (separation by molecular weight from right to left) for the HSP70‚ÄČkDa band, showing that a small fraction of this family of proteins (HSP72, HSP70i or inducible form of HSP70, in the circle) is exclusively expressed in the peri-infarct tissue (PI), while a large fraction of this family (constitutive form of HSP70 or HSP70c) is expressed in both the peri-infarct (PI) and the contra-lateral (CL) tissue. All samples were run together in the same gel.
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f4: Western blot (WB) analysis of the expression of the 70‚ÄČkDa Heat Shock Protein (HSP70) in the ischemic brain.Top) Cropped band of the WB gel corresponding to HSP70 in control tissue (CL, contra-lateral hemisphere), peri-infarct (PI), and the core of the ischemic lesion (L) from the 1D western blot (isoelectric point). All 3 samples were run together in the same gel. ő≤-Actin was used as the loading control (cropped band shown) in the same gel and at the same time (complete gels presented in supplementary data 1). Middle) Quantification of the expression levels, presented as the HSP70/Actin ratio (mean‚ÄȬĪ‚ÄČSDEV of pooled data from two different rats). Bottom) Cropped band corresponding to the second dimension of the western blot study over 2D electrophoresis gels (separation by molecular weight from right to left) for the HSP70‚ÄČkDa band, showing that a small fraction of this family of proteins (HSP72, HSP70i or inducible form of HSP70, in the circle) is exclusively expressed in the peri-infarct tissue (PI), while a large fraction of this family (constitutive form of HSP70 or HSP70c) is expressed in both the peri-infarct (PI) and the contra-lateral (CL) tissue. All samples were run together in the same gel.

Mentions: We performed a conventional Western blot analysis of the expression of the 70‚ÄČkDa family of heat shock proteins from peri-infarct (PI), contra-lateral (CL) and lesion-core (L) tissue samples (Fig. 4, top and supplementary data 1), and separated the proteins according to their molecular weight. ő≤-Actin was used as protein loading control. When the levels of expression of the HSP70 protein were corrected by ő≤-Actin (Fig. 4, middle), we observed no significant differences in the expression between the contra-lateral tissue (HSP70/ő≤-Actin ratio of 5.1) and the peri-infarct tissue (HSP70/ő≤-Actin ratio of 4.8), which were both significantly higher than the expression of HSP70 at the core of the lesion (HSP70/ő≤-Actin ratio of 1.9). Similar results were obtained for other tested proteins that showed significant differences in expression in 2D-PAGE studies, such as dihydropyrimidase-related protein 2 (DHRP-2), eukaryotic translation initiation factor 2 (eIF2őĪ), HSP27, prohibitin and ő≤ -tubulin (data not shown).


Study of Protein Expression in Peri-Infarct Tissue after Cerebral Ischemia.

Brea D, Agulla J, Staes A, Gevaert K, Campos F, Sobrino T, Blanco M, D√°valos A, Castillo J, Ramos-Cabrer P - Sci Rep (2015)

Western blot (WB) analysis of the expression of the 70‚ÄČkDa Heat Shock Protein (HSP70) in the ischemic brain.Top) Cropped band of the WB gel corresponding to HSP70 in control tissue (CL, contra-lateral hemisphere), peri-infarct (PI), and the core of the ischemic lesion (L) from the 1D western blot (isoelectric point). All 3 samples were run together in the same gel. ő≤-Actin was used as the loading control (cropped band shown) in the same gel and at the same time (complete gels presented in supplementary data 1). Middle) Quantification of the expression levels, presented as the HSP70/Actin ratio (mean‚ÄȬĪ‚ÄČSDEV of pooled data from two different rats). Bottom) Cropped band corresponding to the second dimension of the western blot study over 2D electrophoresis gels (separation by molecular weight from right to left) for the HSP70‚ÄČkDa band, showing that a small fraction of this family of proteins (HSP72, HSP70i or inducible form of HSP70, in the circle) is exclusively expressed in the peri-infarct tissue (PI), while a large fraction of this family (constitutive form of HSP70 or HSP70c) is expressed in both the peri-infarct (PI) and the contra-lateral (CL) tissue. All samples were run together in the same gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495553&req=5

f4: Western blot (WB) analysis of the expression of the 70‚ÄČkDa Heat Shock Protein (HSP70) in the ischemic brain.Top) Cropped band of the WB gel corresponding to HSP70 in control tissue (CL, contra-lateral hemisphere), peri-infarct (PI), and the core of the ischemic lesion (L) from the 1D western blot (isoelectric point). All 3 samples were run together in the same gel. ő≤-Actin was used as the loading control (cropped band shown) in the same gel and at the same time (complete gels presented in supplementary data 1). Middle) Quantification of the expression levels, presented as the HSP70/Actin ratio (mean‚ÄȬĪ‚ÄČSDEV of pooled data from two different rats). Bottom) Cropped band corresponding to the second dimension of the western blot study over 2D electrophoresis gels (separation by molecular weight from right to left) for the HSP70‚ÄČkDa band, showing that a small fraction of this family of proteins (HSP72, HSP70i or inducible form of HSP70, in the circle) is exclusively expressed in the peri-infarct tissue (PI), while a large fraction of this family (constitutive form of HSP70 or HSP70c) is expressed in both the peri-infarct (PI) and the contra-lateral (CL) tissue. All samples were run together in the same gel.
Mentions: We performed a conventional Western blot analysis of the expression of the 70‚ÄČkDa family of heat shock proteins from peri-infarct (PI), contra-lateral (CL) and lesion-core (L) tissue samples (Fig. 4, top and supplementary data 1), and separated the proteins according to their molecular weight. ő≤-Actin was used as protein loading control. When the levels of expression of the HSP70 protein were corrected by ő≤-Actin (Fig. 4, middle), we observed no significant differences in the expression between the contra-lateral tissue (HSP70/ő≤-Actin ratio of 5.1) and the peri-infarct tissue (HSP70/ő≤-Actin ratio of 4.8), which were both significantly higher than the expression of HSP70 at the core of the lesion (HSP70/ő≤-Actin ratio of 1.9). Similar results were obtained for other tested proteins that showed significant differences in expression in 2D-PAGE studies, such as dihydropyrimidase-related protein 2 (DHRP-2), eukaryotic translation initiation factor 2 (eIF2őĪ), HSP27, prohibitin and ő≤ -tubulin (data not shown).

Bottom Line: Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue.Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain.Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain.

View Article: PubMed Central - PubMed

Affiliation: 1] Neurology Department, Neurovascular Area, Clinical Neurosciences Research Laboratory, University Clinical Hospital, Health Research Institute of Santiago de Compostela (IDIS), University of Santiago de Compostela, Spain [2] Cellular and Molecular Neurobiology Research Group and Grup de Recerça en Neurociencies del IGTP, Department of Neurosciences, Fundació Institut d'Investigació en Ciències de la Salut Germans Trias I Pujol-Universitat Autónoma de Barcelona, Badalona, Spain.

ABSTRACT
In this work, we report our study of protein expression in rat peri-infarct tissue, 48 h after the induction of permanent focal cerebral ischemia. Two proteomic approaches, gel electrophoresis with mass spectrometry and combined fractional diagonal chromatography (COFRADIC), were performed using tissue samples from the periphery of the induced cerebral ischemic lesions, using tissue from the contra-lateral hemisphere as a control. Several protein spots (3408) were identified by gel electrophoresis, and 11 showed significant differences in expression between peri-infarct and contra-lateral tissues (at least 3-fold, p < 0.05). Using COFRADIC, 5412 proteins were identified, with 72 showing a difference in expression. Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue. Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain. Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain.

No MeSH data available.


Related in: MedlinePlus