Data in support of alpha1beta1 and integrin-linked kinase interact and modulate angiotensin II effects in vascular smooth muscle cells

The data provides information in support of the research article Moraes et al., Atherosclerosis 243(2) (2015) 477–485 [1]. Here we provide data behind the mechanisms involved in Angiotensin II (Ang II) effects on vascular smooth muscle cells (VSMC). Ang II-induced VSMC ROS production is modulated by alpha1beta1 integrin. Ang II also stimulates ROS production in VSMC via p47phox, a NOX2 subunit. Furthermore, Ang II effect on VSMC migration was also inhibited by NOX2 inhibitor. We showed that obtustatin, alpha1beta1 integrin blocker, inhibited Ang II effect on p47phox activation. Ang II effect on ROS production is also PI3K dependent. Finally we showed that NOX1 and Integrin-Linked-Kinase (ILK) are crucial to NOX2 activation. The research provides information about the sequential events of NOX1/alpha1beta1 integrin/ILK/NOX2 in Ang II effects on VSMC.


a b s t r a c t
The data provides information in support of the research article Moraes et al., Atherosclerosis 243(2) (2015) 477-485 [1]. Here we provide data behind the mechanisms involved in Angiotensin II (Ang II) effects on vascular smooth muscle cells (VSMC). Ang IIinduced VSMC ROS production is modulated by alpha1beta1 integrin. Ang II also stimulates ROS production in VSMC via p47 phox , a NOX2 subunit. Furthermore, Ang II effect on VSMC migration was also inhibited by NOX2 inhibitor. We showed that obtustatin, alpha1beta1 integrin blocker, inhibited Ang II effect on p47 phox activation. Ang II effect on ROS production is also PI3K dependent. Finally we showed that NOX1 and Integrin-Linked-Kinase (ILK) are crucial to NOX2 activation. The research provides information about the sequential events of NOX1/alpha1beta1 integrin/ILK/NOX2 in Ang II effects on VSMC.
& The data presented in this article and is related to [1] Value of the data These data provides a thorough understanding the molecular events underlying Angiotensin II effect on vascular smooth muscle cells.
The data shows dual-NOX involvement in Angiotensin II effect on vascular smooth muscle cell: a rapid NOX1 activation which is accompanied by NOX2.
The data shows that Angiotensin II effect on NOX2 activation depends on NOX1 and ILK. These data also show that NOX2 is involved in Angiotensin II effect on vascular smooth muscle cell migration.
1. Data 1.1. Alpha1beta1 modulates the second interval of Ang II-induced ROS production in VSMC Table 1 shows the detailed analysis of ROS production showed in Fig. 1B of Ref. [1]. This table shows that ROS production induced in VSMC occurred in two stages, and that obtustatin inhibited only the second part (0.5-1 h) of ROS production. Table 1 Alpha1beta1 modulates the second interval of Ang II-induced ROS production in VSMC. Slope interval was calculated by Δy/Δx of ROS production graph (see Fig. 1B in Ref. [1]). Δy is the amount of ROS production in determined interval time. Δx is a time interval of 30 min.

Control
Ang II

Time-course of angiotensin II-induced ROS production and its dependence on NOX2 in VSMC
Apocynin, a selective inhibitor of p47 phox (a NOX2 subunit), inhibited only the second peak of ROS (0.5-1 h) production induced by Ang II in VSMC (Fig. 1).

Angiotensin II induces VSMC migration via NOX2
Ang II effect on VSMC migration also relies in NOX2 activity, once we observed that the pretreatment of VSMC with Apocynin inhibited the chemotactic effect of Ang II (Fig. 2).

A7r5 contains NOX1, NOX2 and NOX4
It is well described that VSMC contains NOX2 [2,3], so we investigated the presence of this protein in the VSMC line A7r5. In our data we observed that besides NOX1 and NOX4, A7r5 also has NOX2 at similar levels compared to polymorphonuclear neutrophils (Fig. 3).

p47 phox and alpha1beta1 integrin modulate angiotensin II effect on NOX2 activation in VSMC
When p47 phox interacts with the NOX2 membrane subunit, NOX2 becomes a full complex and it is ready to initiate ROS production [4,5]. Fig. 4 shows that Ang II induced p47 phox translocation to plasma membrane and that apocynin and obtustatin inhibited this effect, indicating a dependence on p47 phox and alpha1beta1 integrin for Ang II-induced NOX2 activation.

Angiotensin II induces p47 phox activation in VSMC
As we can observe through cytosol/membrane fraction analysis, Ang II was able to induce p47 phox translocation to VSMC membrane 10 min after treatment (Fig. 5A). Densitometry can be observed in Fig. 5B.

Angiotensin II induces ROS production via PI3K/AKT in VSMC
The treatment of VSMC with LY294002, a PI3K-AKT inhibitor, impaired the second peak of ROS production (after 30 min) induced by Ang II (Fig. 6).

siRNA silencing in VSMC
For the set of experiments using siRNA technique we used A7r5 rat aorta smooth muscle cell line, which exhibit similar responses to Ang II when compared to primary VSMC (data not shown). Fig. 7A shows the western blotting of whole cell lysate of cells transfected with the specific siRNA (ILK, NOX1 and NOX2) and blotted to ILK, NOX1, NOX2 and actin. Specific siRNA treatment induced, respectively, 50% inhibition in ILK and NOX1 expression and almost 80% reduction of NOX2 (Fig. 7B). It was not possible to completely silence ILK and NOX1 expression since it caused cell death (data not shown).
1.9. ILK modulates the second interval of Ang II-induced ROS production in VSMC Table 2 shows that Ang II was not able to induce any peak of ROS production in A7r5 silenced cells to NOX1. Furthermore silencing to ILK just interfered with the second peak of ROS production induced by Ang II.

Alpha1beta1 integrin does not modulate Angiotensin II effect on mitochondrial ROS production in VSMC
Ang II induced mitochondrial ROS production in VSMC after 3 h of treatment. We also observed that this effect was not alpha1beta1 integrin dependent, once obtustatin was not able to inhibit Ang II effect (Fig. 9). . siRNA silencing in VSMC. A7r5 cells were transfected with siRNA to NOX1, NOX, ILK or siRNA scramble for 24 h. All treatments were performed at 37°C/5% CO 2 . A. Whole cell extracts were prepared and subjected to Western blotting for ILK, NOX1, NOX2 and β-actin (load control). B. Effect of each siRNA on its respective target was quantified and it was showed in % of silencing. Table 2 ILK modulates the second interval of Ang II-induced ROS production in VSMC. Slope interval was calculated by Δy/Δx of ROS production graph (see Figure 3B in Ref. [1]). Δy is the amount of ROS production in determined interval time. Δx is a time interval of 30 min.

Control
Ang II

Proposed model
Taking together this data and Ref. [1], Ang II modulates signaling pathways involved in chemotaxis and proliferation in vascular smooth muscle cells. Ang II induces a rapid ROS production via NOX1. This initial and rapid ROS production activates alpha1beta1 integrin, which in turn activates FAK. Once activated, FAK dissociates to ILK, which activates AKT and induces p21 degradation. AKT promotes p47 phox engagement do NOX2 membrane subunit, allowing NOX2 complex activation, leading to ROS production that is responsible for cell migration and partially to cell proliferation (Fig. 10).

Cell culture
Primary VSMC were isolated from the aortic arch from naïve Wistar male rats using an explant method. In brief, rats were anesthetized and the thoracic aorta was harvested, opened longitudinally, scraped free of endothelium, cut into small pieces and treated for 30 min with collagenase. The endothelium-denuded tissue was placed with the luminal surface facing down on a 0.1% gelatincoated flask with DMEM medium containing 10% FBS, 50 U/mL penicillin and 100 mg/mL streptomycin, and cultured at 37°C/ 5% CO 2 air atmosphere. After 7-14 days, the VSMC that outgrowth from the explants were trypsinized and used in different assays until passage three. These cells were positive stained for VSMC α-actin. This study was carried out in strict accordance with the recom- A7r5 VSMC, obtained from rat thoracic aorta (ATCC, Rockville, MD) were cultured in DMEM medium containing 10% FBS, 50 U/mL penicillin and 100 mg/mL streptomycin, and incubated at 37°C in a 5% CO2 air atmosphere. The cells were dissociated with 0.1%/0.01% trypsin/EDTA and then seeded onto new culture flasks for a maximum of twelve passages. Fig. 10. Proposed model. Ang II-induced ROS production is NOXs dependent and the initial ROS production induced by NOX1 leads to NOX2 activation, which sustains the effect. Alpha1beta1 integrin/ILK crosstalk is a link between NOX1 and NOX2 activation, which in turn is responsible for Ang II-induced VSMC migration and proliferation.

mRNA silencing
A7r5 VSMC were transfected with either a non-targeting control siRNA (siRNA negative control 1; Ambion, TX, USA) as a control for non-sequence-specific effects, or with an ILK (Silencer Pre designed siRNA ILK ID 16708; Ambion), a NOX1-specific siRNA sequence (Silencer Predesigned siRNA NOX1 ID 55585; Ambion) or a NOX2-specific siRNA sequence (Silencer Predesigned siRNA NOX2 ID 647047; Ambion). Twenty mM of either the non-targeting or ILK, NOX1 or NOX2-specific duplex were diluted in 1 mL Opti-MEM I (Gibco,Grand Island, NY, USA) reduced serum medium and 1 mL of Lipofectamine 2000 (Ambion) diluted in Opti-MEM I was added. The mixture was gently mixed and allowed to incubate for 5 min at room temperature, being the final concentration of siRNA 10 mM. VSMC were grown in 6-well plate culture and when at 70% of confluence, RNAi duplex-Lipofectamine 2000 complexes in antibiotics and serum-free media was added. Twenty-four hours after transfection, attenuation of ILK, NOX1 or NOX2 expression was verified by Western blotting of cell lysates probed with antibody against ILK, NOX1 and NOX2.

Reactive oxygen species production
VSMC (5 Â 10³ cells/well) were seeded in 96 wells plate overnight in DMEM medium containing 10% FBS. The cells were washed three times with PBS and the medium was replaced by DMEM containing 1% FCS for 1 h. VSMC were loaded with 10 mM CM-H2DCFDA (intracellular ROS) or with 10 mM MitoSox Red (mitochondrial-derived ROS) for 1 h and then washed to remove free probe. Cells were pretreated with 100 nM obtustatin or 10 mM apocynin for 15 min, and then incubated with Ang II 100 nM for 3 h at 37°C. CM-H2DCFDA fluorescence was monitored at excitation and emission wavelengths of 495 and 530 nm, respectively and MitoSox Red fluorescence was monitored at excitation and emission wavelengths of 510 and 580 nm, respectively. Fluorescence was quantified using an EnVision s multilabel plate reader (Perkin-Elmer, Waltham, MA, USA). Slope intervals showed in tables were calculated by Δy/Δx of ROS production graph. Δy is the amount of ROS production in determined interval time. Δx is a time interval of 30 min.

Cell migration assay
For cell migration (chemotaxis) analysis in vitro, VSMC were seeded at a density of 2 Â 105 cells/ well in 48-well modified Boyden chambers containing a polycarbonate membrane (pore size, 10 mm; Neuroprobe, Inc., Gaithersburg, MD, USA), and incubated at 37°C in a 5% CO 2 atmosphere for 4 h. Some cell samples were treated with 10 mM Apocynin for 15 min before addition to the upper chamber (50 mL), and VSMC migrated toward 100 nM Ang II (in lower chamber). The cells that migrated to the lower membrane surface were fixed and stained using Wright-Giemsa stain and counted by light microscopy (400 Â ), in an Olympus BX41 light microscope (Tokyo, Japan).