Journal: Physiological Reports

Article Title: Hypoxic pulmonary endothelial cells release epidermal growth factor leading to vascular smooth muscle cell arginase‐2 expression and proliferation

doi: 10.14814/phy2.15342

Figure Lengend Snippet: Conditioned media from hPMVEC incubated in hypoxia induced Arg2 protein expression and proliferation in normoxic hPASMC. Conditioned media was generated from hPMVEC incubated in normoxia (NCM) or hypoxia (HCM) for 24 h. The NCM or HCM was then placed on hPASMC with smooth muscle media in a 50:50 ratio and the hPASMC were incubated in room air with 5% CO 2 for 24 h. (a) Protein was harvested for western blotting for Arg2 and β‐actin. Representative western blot images. (b) Arg2 protein levels were greater in hPASMC incubated with HCM than in those incubated with NCM. Densitometry for the Arg2 western blots normalized to β‐actin. *Different from NCM p < 0.003. n = 3 for each group. (c) To measure viable hPASMC numbers, 1 × 10 4 hPASMC were plated in each well of 6‐well plates, NCM or HCM were placed on the cells, and after incubating in normoxia for 48 h viable cell numbers were determined using trypan blue exclusion. Viable hPASMC numbers were significantly greater after incubation with HCM than after incubation with NCM. *Ddifferent from NCM p < 0.0001. n = 6 for each group. (d) To determine if conditioned media from hypoxic hPASMC would stimulate an increase in hPMVEC viable cell numbers, conditioned media was generated from hPASMC incubated for 24 h in either 20% O 2 /5% CO 2 (SMC‐NCM) or 1% O 2 /5% CO 2 (SMC‐HCM). Human PMVEC (1 × 10 4 ) were plated in each well of 6 well plates, the SMC‐NCM or SMC‐HCM were placed on the hPMVEC , and after 48 h viable cell number determined using trypan blue exclusion. There was no difference ( p = 0.33) in viable endothelial cell numbers between hPMVEC incubated in SMC‐HCM versus hPMVEC incubated in SMC‐NCM. n = 6 for each group.

Article Snippet: The hPASMC were cultured in SMC media (ScienCell, Carlsbad, CA) supplemented with a SMC kit containing fetal bovine serum, SMC growth supplement, and a penicillin/streptomycin solution (ScienCell).

Techniques: Incubation, Expressing, Generated, Western Blot

Journal: Physiological Reports

Article Title: Hypoxic pulmonary endothelial cells release epidermal growth factor leading to vascular smooth muscle cell arginase‐2 expression and proliferation

doi: 10.14814/phy2.15342

Figure Lengend Snippet: Conditioned media from hPMVEC incubated in hypoxia induced Arg2 protein expression and proliferation in normoxic hPASMC. Conditioned media was generated from hPMVEC incubated in normoxia (NCM) or hypoxia (HCM) for 24 h. The NCM or HCM was then placed on hPASMC with smooth muscle media in a 50:50 ratio and the hPASMC were incubated in room air with 5% CO 2 for 24 h. (a) Protein was harvested for western blotting for Arg2 and β‐actin. Representative western blot images. (b) Arg2 protein levels were greater in hPASMC incubated with HCM than in those incubated with NCM. Densitometry for the Arg2 western blots normalized to β‐actin. *Different from NCM p < 0.003. n = 3 for each group. (c) To measure viable hPASMC numbers, 1 × 10 4 hPASMC were plated in each well of 6‐well plates, NCM or HCM were placed on the cells, and after incubating in normoxia for 48 h viable cell numbers were determined using trypan blue exclusion. Viable hPASMC numbers were significantly greater after incubation with HCM than after incubation with NCM. *Ddifferent from NCM p < 0.0001. n = 6 for each group. (d) To determine if conditioned media from hypoxic hPASMC would stimulate an increase in hPMVEC viable cell numbers, conditioned media was generated from hPASMC incubated for 24 h in either 20% O 2 /5% CO 2 (SMC‐NCM) or 1% O 2 /5% CO 2 (SMC‐HCM). Human PMVEC (1 × 10 4 ) were plated in each well of 6 well plates, the SMC‐NCM or SMC‐HCM were placed on the hPMVEC , and after 48 h viable cell number determined using trypan blue exclusion. There was no difference ( p = 0.33) in viable endothelial cell numbers between hPMVEC incubated in SMC‐HCM versus hPMVEC incubated in SMC‐NCM. n = 6 for each group.

Article Snippet: The hPASMC were cultured in SMC media (ScienCell, Carlsbad, CA) supplemented with a SMC kit containing fetal bovine serum, SMC growth supplement, and a penicillin/streptomycin solution (ScienCell).

Techniques: Incubation, Expressing, Generated, Western Blot