Thomas E. Arnold, MD, Dmitri Gnatenko, PhD, Wadie F. Bahou, MD

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Presentation transcript:

In vivo gene transfer into rat arterial walls with novel adeno-associated virus vectors Thomas E. Arnold, MD, Dmitri Gnatenko, PhD, Wadie F. Bahou, MD Journal of Vascular Surgery Volume 25, Issue 2, Pages 347-355 (February 1997) DOI: 10.1016/S0741-5214(97)70357-4 Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Microscopic views of sections from rat carotid arteries (original magnification ×40). All vessels were stained by DNA in situ polymerase chain reaction (PCR) to detect the lacZ gene, except for panels E and F. Lu indicates the vessel lumen. EC and SMC indicate endothelial cell and smooth muscle cell, respectively. A through D, Show vessels harvested 24 hours after instillation of the solution indicated to the left of each photomicrograph. A and B, Show arteries transduced with individual recombinant adeno-associated virus (rAAV) vector solutions. Dark blue staining of cell nuclei provides evidence for the presence of the lacZ gene. C, Shows an artery instilled with normal saline solution. D, Shows an artery that has not been operated on. In panels C and D, no blue staining is present (nuclear fast red stain). E, Shows a transduced artery stained by DNA in situ PCR to detect endogenous rat β-actin, to serve as a positive control. F, Shows an artery transduced with rAAV/Col and stained with hematoxylin and eosin stain. No significant cell loss or inflammatory infiltrates are present. G and H, Show DNA in situ PCR of arteries 1 week and 2 months after gene transfer with rAAV/Col, respectively. The percentage of cell nuclei that contain the lacZ gene decreases with time. At 2 months, lacZ is present in nuclei near the vessel lumen (nuclear fast red stain). Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Graph showing the level of β-galactosidase expression at the interval indicated after in vivo gene transfer. The vector instilled is indicated to the left of each pair of bars. Bars with plus signs (+) represent arteries instilled with a vector solution; bars with circles ( ̂) represent paired unoperated control arteries. Activity is expressed as microunits per milligram of total protein in the tissue extract. Bars indicate the mean β-galactosidase activity in each group. Each data point represents a single vessel. For each group, r represents the mean of the ratios that compare activity in the instilled versus the paired unoperated artery for each individual animal in that group (see text). Where p values are absent, the sample size (n) was too small to allow statistical analysis. Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Graphs showing β-galactosidase antigen at time intervals of 24 hours and 1 week (A) and β-galactosidase activity at varying virus concentrations (B). Solid bars represent arteries incubated with rAAV/Col vector solution. Open bars represent paired unoperated control arteries. Expression is expressed as picograms (A) or microunits (B) per milligram of total protein in the tissue extract. Bars indicate the mean β-galactosidase expression in each group. Each data point represents a single vessel. For each group in graph B, r represents the mean of the ratios that compare activity in the instilled versus the paired unoperated artery for each individual animal in that group (see text). Where p values are absent, the sample size (n) was too small to allow statistical analysis. iu, Infectious units. Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Graphs showing β-galactosidase antigen at time intervals of 24 hours and 1 week (A) and β-galactosidase activity at varying virus concentrations (B). Solid bars represent arteries incubated with rAAV/Col vector solution. Open bars represent paired unoperated control arteries. Expression is expressed as picograms (A) or microunits (B) per milligram of total protein in the tissue extract. Bars indicate the mean β-galactosidase expression in each group. Each data point represents a single vessel. For each group in graph B, r represents the mean of the ratios that compare activity in the instilled versus the paired unoperated artery for each individual animal in that group (see text). Where p values are absent, the sample size (n) was too small to allow statistical analysis. iu, Infectious units. Journal of Vascular Surgery 1997 25, 347-355DOI: (10.1016/S0741-5214(97)70357-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions