Nitric oxide synthase inhibitors attenuate transforming-growth-factor- β1-stimulated capillary organization in vitro

Andreas Papapetropoulos, Kaushik M. Desai, R. Daniel Rudic, Bernd Mayer, Rong Zhang, Maria P. Ruiz-Torres, Guillermo García-Cardeña, Joseph A. Madri, William C. Sessa

Research output: Contribution to journalArticle

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Abstract

Angiogenesis is a complex process involving endothelial celt (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-β 1 . Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-β 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs.

Original languageEnglish (US)
Pages (from-to)1835-1844
Number of pages10
JournalAmerican Journal of Pathology
Volume150
Issue number5
StatePublished - May 17 1997
Externally publishedYes

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Transforming Growth Factors
Nitric Oxide Synthase
NG-Nitroarginine Methyl Ester
Nitric Oxide
Arginine
Protein Isoforms
Nitric Oxide Donors
Nitroprusside
Nitrites
Reverse Transcriptase Polymerase Chain Reaction
Collagen
Western Blotting
Gels
In Vitro Techniques
Proteins

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Papapetropoulos, A., Desai, K. M., Rudic, R. D., Mayer, B., Zhang, R., Ruiz-Torres, M. P., ... Sessa, W. C. (1997). Nitric oxide synthase inhibitors attenuate transforming-growth-factor- β1-stimulated capillary organization in vitro. American Journal of Pathology, 150(5), 1835-1844.

Nitric oxide synthase inhibitors attenuate transforming-growth-factor- β1-stimulated capillary organization in vitro. / Papapetropoulos, Andreas; Desai, Kaushik M.; Rudic, R. Daniel; Mayer, Bernd; Zhang, Rong; Ruiz-Torres, Maria P.; García-Cardeña, Guillermo; Madri, Joseph A.; Sessa, William C.

In: American Journal of Pathology, Vol. 150, No. 5, 17.05.1997, p. 1835-1844.

Research output: Contribution to journalArticle

Papapetropoulos, A, Desai, KM, Rudic, RD, Mayer, B, Zhang, R, Ruiz-Torres, MP, García-Cardeña, G, Madri, JA & Sessa, WC 1997, 'Nitric oxide synthase inhibitors attenuate transforming-growth-factor- β1-stimulated capillary organization in vitro', American Journal of Pathology, vol. 150, no. 5, pp. 1835-1844.
Papapetropoulos, Andreas ; Desai, Kaushik M. ; Rudic, R. Daniel ; Mayer, Bernd ; Zhang, Rong ; Ruiz-Torres, Maria P. ; García-Cardeña, Guillermo ; Madri, Joseph A. ; Sessa, William C. / Nitric oxide synthase inhibitors attenuate transforming-growth-factor- β1-stimulated capillary organization in vitro. In: American Journal of Pathology. 1997 ; Vol. 150, No. 5. pp. 1835-1844.
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abstract = "Angiogenesis is a complex process involving endothelial celt (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-β 1 . Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50{\%}. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-β 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs.",
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AU - Papapetropoulos, Andreas

AU - Desai, Kaushik M.

AU - Rudic, R. Daniel

AU - Mayer, Bernd

AU - Zhang, Rong

AU - Ruiz-Torres, Maria P.

AU - García-Cardeña, Guillermo

AU - Madri, Joseph A.

AU - Sessa, William C.

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N2 - Angiogenesis is a complex process involving endothelial celt (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-β 1 . Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-β 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs.

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