Nano Encapsulated Transforming Growth Factor-β (TGF-β) Possibly Attenuates NF-κB Activation Pathway in Pulmonary Hypertension

Leandro Fernandez, Omar Mohtar, Elizabeth Klings, and Jean-Bosco TagnePulmonary Center, Boston University Medical Center

Introduction• Pulmonary hypertension (PH) is a clinical condition resulting from pulmonary vasculopathy. The epidemiologic

associations between PH and systemic hypertension, renal and cerebrovascular disease in sickle cell disease (SCD) suggests a pathologic link amongst vascular complications of SCD.

• Candidate gene-based studies done by our group and others have demonstrated that Transforming Growth Factor-Beta (TGF-β), a secreted protein in the superfamily of cytokines that performs many cellular functions is associated with numerous vascular phenotypes in sickle cell disease including stroke, priapism, leg ulcerations and pulmonary hypertension.

• These functions include the control of cell growth, cell proliferation, as well as cell differentiation and apoptosis. As a result of TGF-β’s wide array of cellular processes, it can be negatively or positively expressed to induce or inhibit these cellular functions.

• Aberrant TGF-β signaling has roles in animal models of PH and mutations have been found in some patients with idiopathic PH suggesting that this pathway is an important modulator of gene expression within the pulmonary vasculature.

Hypothesis

Human pulmonary artery endothelial (HPAEC) cells were kindly provided by Dr. Jean-Bosco Tagne (Pulmonary Center, Boston University Medical Center) and were grown in p100 plates with Lonza’s EGM™-2 BulletKit™ culture media. The media was changed every three days for continued cell growth. The experiment with these cells began when they reached 70% confluence. Each well was then added with one of the following treatments: 5μM TGF-β, 10μM TGF-β, 5nM nano-formulated TGF-β, 10 nM nano-formulated TGF-β, or left untreated as a control. Cells were incubated for 48 hours and then harvested for RT-PCR analysis. Real-time reverse transcription polymerase chain reaction (RT- PCR) methods were adapted to identify the different genes of interest. Expression levels of Smad 3 for its role in the TGF-β signaling pathway, Map3k7, as well as RelA, both of which are fundamental for the Nuclear Factor NF-κB pathway in TGF-β, and Fibronectin (FN1) which is responsible for cell adhesion, wound healing, and cell migration were analyzed using real-time quantitative PCR as a validation of microarray results.

Methods

All nanoparticle samples were measured for radius in nanometers immediately after preparation showing a profile of under 200 nanometers that peaked at about 166 nanometers.

A B C

(A) HPAEC cells treated with TGF-β after 48 hours of incubation while (B) shows cells treated with the nano-formulated TGF-β at the same length of time. (C) untreated HPAEC cells

We hypothesize that aberrant TGF-β signaling within the endothelium may play a role in disease modulation in SCD patients with PH. We plan to address the question of whether direct genetic targeting of the endothelium in SCD can alter these interactions and potentially, impact the development of vasculopathy.

RESULTS

CONCLUSIONS

REFERENCES1. Tagne, J.B., et al., A nanoemulsion formulation of tamoxifen increases its efficacy in a breast cancer cell line. Mol Pharm, 2008. 5(2): p. 280-62. Freudlsperger, C., et al., TGF-b and NF-kB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers. Oncogene, 2013. 32, 1549–15593. Sultana, Chand, et al., Interaction of Sickle Erythrocytes With Endothelial Cells in the Presence of Endothelial Cell Conditioned Medium Induces Oxidant Stress Leading to Transendothelial Migration of Monocytes Blood, 1998. Vol 92, No 10 (November 15),: pp 3924-3935

• Although concentrations of TGF-β doubled, there was no significance between the two amounts in terms of gene expression and both concentrations follow the same trend.

• The two genes that are in the NF-κb pathway, RelA and Map3k7, both decreased in expression in the nano-TGF-β compared to the regular TGF-β

• Smad 3 which naturally occurs in the TGF-β signaling pathway, increased in nano-formulated TGF-β compared to normal TGF-β

• Fibronectin saw the biggest difference between nano and regular TGF-β with a 1.5 fold decrease in the nano-formulation.

The similar trends between the two varying concentrations suggests that a lower amount of TGF-β is more beneficial because the lesser sum will undeniably have less side effects, if any. The abnormal adherence of sickle red blood cells to endothelial cells has been thought to contribute to vascular occlusion. This, along with transcription factor NF-kB, an indicator of cellular oxidant stress, is a major cause of morbidity in sickle cell disease. This study showed that in the presence of our Nano-formulated TGF-β, the nuclear factor NF-κB pathway is altered, and an important gene in regulating cell adhesion (FN1) is also considerably lower which is an indication of the improvement in TGF-β’s therapeutic effect in the cure of PH and SCD.

Nano Encapsulated Transforming Growth Factor-β (TGF-β) Possibly Attenuates NF-κB Activation Pathway in Pulmonary Hypertension

Pulmonary Center, Boston University Medical Center

Leandro Fernandez, Omar Mohtar, Elizabeth Klings, and Jean-Bosco Tagne

The figure shown above shows the RT-PCR results for the 4 different genes of interest using delta delta ct comparative analysis. The blue bars indicate the normal TGF-β while the red bars show the expression for the nano-TGF-β. The graphs are also placed side by side to their 5 and 10 molar counterparts to further illustrate the trend between the two concentrations

FN1 FN1TGF 10 nM NTGF 10 nM

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FN1 FN1TGF 5nm NTGF 5nm

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MAP3K7 MAP3K7TGF 5nm NTGF 5nm

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1.1 5uM Map3k7 Relative Expression

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MAP3K7 MAP3K7TGF 10 nM NTGF 10 nM

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RELA RELATGF 10 nM NTGF 10 nM

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1.2 10uM RelA Relative Ex-pression

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Smad3 Smad3TGF 5nm NTGF 5nm

0.7

0.72

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0.78

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0.82

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0.88 5uM Smad 3 Relative Ex-pression

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SMAD3 SMAD3TGF 10 nM NTGF 10 nM

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0.910uM Smad 3 Relative

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