Clone 1a4

Primary antibodies used for the studies included: (1) A mouse monoclonal antibody to α‐smooth muscle actin (Durand‐Arczynska et al., 1993 (link)) (clone 1A4, catalog number: A5228, Sigma‐Aldrich), (2) A mouse monoclonal antibody to actin (Lessard, 1988 (link)) (clone C4, catalog number: MA1501, Sigma‐Aldrich), (3) Goat polyclonal antibodies to neprilysin (Sagare et al., 2013 (link)) (catalog number: AF1126) and nephrin (Wong et al., 2018 (link)) (catalog number: AF3159), all from R&D Systems (Minneapolis, MN), and (4) A mouse monoclonal antibody to NPRC (clone OTI4H1, catalog number: TA501044, Origene Technologies). Secondary antibodies used for the studies included: (1) A mouse anti‐goat polyclonal antibody (catalog number: sc‐2354, Santa Cruz Biotechnology), and (2) An anti‐mouse polyclonal antibody (catalog number: 7076, Cell Signaling Technology). Additional materials included: ANP(4‐23) (GenScript), human ANP, human CNP and PF‐04449743 (Sigma‐Aldrich), AP‐811 (Tocris Bioscience), and LBQ657 and tadalafil (Cayman Chemical).

Vascular morphometric analysis in mouse lung sections was performed according to our previously described method.^{19 (link)} Degree of vessel muscularization was assessed by two color IHF staining of lung sections with an anti-αSMA antibody (dilution 1:400, clone 1A4, Sigma-Aldrich Corp., St. Louis, MO, USA) and with an antibody targeting human von Willebrand factor (dilution 1:100, Abcam, Cambridge, MA, USA). IHF-labeled lung sections were scanned with Aperio ScanScope FL at 20 × magnification. To assess the degree of muscularization, vessels (20–100 µm in diameter) were categorized as partially muscularized (< 75% of the vessel circumference immunopositive for anti-αSMA antibody) or fully muscularized (> 75% of the vessel wall immunoreactive against anti-αSMA antibody) as previously described.^{17 (link)} Vessel wall thickness was assessed in vessels immunopositive for αSMA (20–100 µm in diameter; n = 80–100 vessels) for each animal (n = 4/group). Vessel wall thickness was measured using Aperio ImageScope software and calculated using the following equation: Wall thickness = Wall width/Vessel width.

HASMCs were analyzed using monoclonal (Santa Cruz Biotechnology; catalogue number sc‐377218, clone F1) or polyclonal (Santa Cruz Biotechnology; catalogue number sc‐8316, H‐181) antibodies against TrkB or the monoclonal anti–pan‐Trk antibody (Abcam; catalogue number ab181560, clone EPR17341) or monoclonal antibodies against SMA (Sigma Aldrich, clone 1A4) followed by fluorescence‐conjugated secondary antibodies (Alexa Fluor 488, MoBiTec). Cells were washed and analyzed on a FACSCanto I (BD Biosciences). Results are expressed as percent positive cells per 1×10⁶ total cells.

Prior to immunofluorescent stainings, the hydrogel constructs were
permeabilized with 0.2% triton-X in PBS for 30 min and blocked in 5% BSA/PBS for
30 min. Capillarylike structures in the hydrogels were investigated by CD31
staining (5.1 μg mL⁻¹, M0823, Dako), secondary sheep antimouse
biotinylated antibody (1:200, RPN1001v1, GE Healthcare), and tertiary
streptavidin Alexa Fluor 488 conjugate (5.0 μg mL⁻¹, S32354,
Invitrogen). In vasculogenic cocultures, ECFCs with the GFP label were not
stained for CD31. The endothelial phenotype was confirmed by a rabbit
antivascular endothelial cadherin antibody (VE-cad, 1:250, D87F2, Cell
Signalling Technology) which was combined with a secondary donkey-antirabbit
Alexa 647 antibody (5 μg mL⁻¹, ab150075, Abcam). Stabilizing cells of
the capillary-like structures were identified by a mouse monoclonal
Cy3-conjugated aSMA antibody (1:300 μg mL⁻¹, Clone 1A4, C6198 Sigma
Aldrich). Furthermore, 4,6-diamidino-2-phenylindole (DAPI, 100 ng
mL⁻¹, Sigma) was used to stain cell nuclei. The hydrogels were
imaged with a confocal microscope (SP8x Leica, DMi8, Leica).