Research ArticlePulmonary Hypertension

Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension

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Science Translational Medicine  07 Oct 2015:
Vol. 7, Issue 308, pp. 308ra159
DOI: 10.1126/scitranslmed.aaa9712
  • Fig. 1. Hypoxia-induced SMCs in distal pulmonary arterioles derive from a single preexisting SMC.

    SMA-CreERT2, ROSA26R(Rb/+) mice were induced with tamoxifen (1 mg/day for 5 days), rested for 5 days, and then exposed to normoxia or hypoxia (FiO2 10%). (A) Potential patterns of Rb colors in pulmonary arteriole SMCs. Middle (M) arteriole SMCs are present under normoxia and thus marked by different Rb colors [Cerulean (Cer), mOrange (mOr), or mCherry (mCh)]. Hypoxia-induced distal (D) arteriole SMCs will be multiple colors if they derive from multiple polyclonal preexisting SMCs or one color if they derive from a single preexisting SMC. (B) Vibratome sections of lungs isolated from one normoxic mouse and three mice exposed to hypoxia for 7 or 21 days as indicated and imaged with direct fluorescence of Rb color channels. Alveolar SMCs are marked by arrowheads. Scale bar, 25 μm. (C) Quantification of SMCs in the proximal (P), middle (M), or distal (D) arterioles by color. Data are averages ± SD [n = 4 lungs, two to three arterioles per lung; number of cells scored per arteriole by arteriole type were as follows: proximal (each condition, 165 to 230 cells), middle (each condition, 140 to 206), and distal (normoxia, 0; hypoxia, 70 to 108)].

  • Fig. 2. Primed cells are the major source of hypoxia-induced distal arteriole SMCs.

    (A and B) In wild-type (WT) mice, arterioles in proximity to airway branches L.L1.A1 (left bronchus–first lateral secondary branch–first anterior branch) were stained for SMA and PDGFR-β as well as for mouse pan−endothelial cell antigen-32 (MECA-32) [endothelial cell (EC) marker] (A) or SMMHC (B), as indicated. (C) PDGFR-β–CreERT2, ROSA26R(mTmG/+) mice were induced with tamoxifen and rested for 2 days. After the rest period, lungs were stained for PDGFR-β, green fluorescent protein (GFP) lineage tag, and SMMHC. (D to G) PDGFR-β–CreERT2, ROSA26R(mTmG/+) mice were induced with tamoxifen and rested for 5 days. Mice were then exposed to 21 days of normoxia or hypoxia before staining lungs for GFP, SMA, and MECA. The boxed regions in (D) and (F) are shown as close-ups in (E) and (G), and further magnifications of the boxes in (E) are displayed as insets. Results are representative of three mice and two to three arterioles per mouse. Scale bars, 25 μm.

  • Fig. 3. KLF4 is up-regulated with PA SMC dedifferentiation and proliferation in PH and PAH patients.

    (A to C) Paraffin sections of pulmonary arterioles from human controls and PH and PAH patients, which are representative of three control patients (normal lungs and no cardiopulmonary illness), three PH patients (WHO PH group 3, idiopathic pulmonary fibrosis and alveolar hypoventilation/sleep apnea; group 5, sarcoidosis), and two PAH patients (group 1, idiopathic PAH and connective tissue disorder). Sections were stained for KLF4, SMA, nuclei [4′,6-diamidino-2-phenylindole (DAPI)], and either PDGFR-β (A), von Willebrand factor (vWF) (B), or the proliferation marker Ki67 (C). Arrowheads in (C) indicate KLF4+Ki67+ SMCs. Scale bars, 25 μm. (D to G) Quantification from images as shown in (A) to (C), indicating the percent of SMCs that are KLF4+ (D) or Ki67+ (E) as well as the percent of KLF4+ SMCs (F) or Ki67+ SMCs (G) that also express the other marker. Per each patient classification, at least 384 arterioles were scored (D to G); *P < 0.01 versus control. (H and I) Primary human PA SMCs were treated with normoxia or hypoxia (1% O2) for indicated times, and KLF4 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein levels were assayed by Western blot with densitometric analysis; n = 4 distinct samples in duplicate. Data are averages [relative to GAPDH in (I)] ± SD. Single-factor analysis of variance (ANOVA) was used in (D) to (G), and two-factor ANOVA was used in (I).

  • Fig. 4. Mice exposed to hypoxia have enhanced KLF4 expression in pulmonary arteriole SMCs.

    Mice were exposed to normoxia or indicated days of hypoxia (FiO2 10%), and then arterioles in proximity to L.L1.A1 airway branches were analyzed. (A) Lung vibratome sections stained for KLF4, SMA, MECA32, and nuclei (DAPI). (B) Quantification of KLF4+ SMCs in proximal (>75 μm diameter), middle (25 to 75 μm), and distal (<25 μm) arterioles. The middle arterioles were further classified into proximal (Ma) and distal (Mb) subdivisions (Materials and Methods). Images are representative of n = 4 lungs, two to three arterioles per lung; number of SMA+ cells scored per arteriole by arteriole type were as follows: proximal (each condition, 170 to 220 SMA+ cells), Ma (each condition, 100 to 120), Mb (each condition, 50 to 65), or distal (normoxia, 0; hypoxia 3 days, 25; hypoxia 7 days, 42; hypoxia 21 days, 110). *P < 0.001 versus normoxia, distal arterioles; ^P < 0.01 versus normoxia middle arteriole, Mb subdivision. (C) Arterioles from mice at normoxia or hypoxia days 2 and 7 stained for SMA, PDGFR-β, and KLF4. Images are representative of 13 arterioles. (D) Quantification of KLF4+ SMCs in proximity to M-D border (Mb region) at hypoxia day 2. Data are averages ± SD (n = 5 lungs, two to three arterioles per lung; total KLF4+ cells were 43). Statistical tests used were two-factor ANOVA (B) and Student’s t test (D). Scale bars, 25 μm.

  • Fig. 5. KLF4 is required cell autonomously in SMCs for distal pulmonary arteriole muscularization and PH.

    (A to F) Mice were injected with tamoxifen [1 mg/day for 5 days in (A) to (E) for complete Klf4 deletion or a single 1-mg injection in (F) for mosaic analysis], rested for 5 days (A to E) or 3 days (F), and then exposed to normoxia or hypoxia (FiO2 10%) for 3 days (D) or 21 days (A to C, E, and F). In SMA-CreERT2, Klf4(flox/flox) mice, arterioles in proximity to L.L1.A1 (A and E) or L.M1 (left bronchus–first medial branch) (D) airway branches were stained for SMA and MECA-32 and also for PDGFR-β (D and E) as indicated. Arrowheads in (D) indicate primed cells that migrated beyond the M-D border. Measurements of RV systolic pressure [RVSP; equivalent to PA systolic pressure] and the ratio of the weight of the RV to that of the sum of the left ventricle (LV) and septum (S) are shown (B and C). Data are averages ± SD (n = 4 mice). Statistical test used in (B) and (C) was two-factor ANOVA. Distal arterioles of the L.L1.A1 regions in SMA-CreERT2, Klf4(flox/flox), ROSA26R(mTmG/+) mice were stained for SMA, GFP lineage tag, and MECA32 (for all immunohistochemistry experiments; n = 4 lungs for each condition and two to three arterioles per lung). Scale bars, 25 μm.

  • Fig. 6. Hypoxia induces polarized PDGF-B lung expression, and reduced PDGF-B prevents distal arteriole muscularization, PH, and primed cell KLF4 expression.

    (A) Adult WT mice were exposed to normoxia or to 2, 7, or 21 days of hypoxia (FiO2 10%), and for each time point, coronal left lung sections are shown as large panels on the left with staining for PDGF-B and SMA. Arrows indicate boxed regions, which contain an arteriole in proximity to the L.L1.A1.M1 airway and are displayed as two close-ups (without or with staining for MECA-32) on the right for each time point (n = 3 lungs, two to three arterioles per lung). (B to D) PDGF-B(+/−) or WT mice were exposed to normoxia or hypoxia for 21 days, and then RVSP and ventricle mass ratio were measured, and left lungs were stained for PDGF-B, SMA, and MECA-32. Data in (C) and (D) are averages ± SD. [n = 4 lungs for (B) to (D), and two to three arterioles were stained as in (B) per lung]. (E) The M-D border of arterioles in proximity to the L.M1 airway is shown for WT and PDGF-B(+/−) mice exposed to hypoxia for 2 days. Sections were stained for KLF4, SMA, and PDGFR-β. (F) Quantification of the percentage of primed cells that express KLF4 from images, such as those shown in (E), of hypoxic WT and PDGF-B(+/−) mice. Data are averages ± SD [n = 4 lungs, two to three arterioles per lung, and total scored primed cells were 28 in WT and 32 in PDGF-B(+/−)]. Statistical tests used were two-factor ANOVA (C and D) and Student’s t test (F). Scale bars, 20 mm (A) and 25 μm [(B) and (E)].

  • Fig. 7. Summary of molecular and cellular events in hypoxia-induced distal pulmonary arteriole muscularization.

    (A) Under normoxic conditions, SMCs (red outline) coat proximal and middle, but not distal, pulmonary arteriole EC tubes and express SMA and SMMHC (no fill). In addition, rare PDGFR-β+SMA+SMMHC+ primed SMCs (pink fill) are located at each M-D border, which coincides with the transition from muscularized to unmuscularized arteriole. (B) Upon initial hypoxic exposure, lung PDGF-B expression is markedly increased, which is required for KLF4 induction in primed cells (pink fill with red dot). (C) Within a day after KLF4 expression, an induced primed SMC (a KLF4+PDGFR-β+SMA+SMMHC+ cell) migrates distally across the M-D border and, on the basis of our previous work (10), dedifferentiates, as indicated by down-regulating SMMHC expression (yellow fill with red dot). (D) Subsequently, the dedifferentiated cell clonally expands, giving rise the vast majority of distal arteriole SMCs. (E) These cells then reexpress SMMHC (10), and again, rare primed SMCs are localized at the now distally located muscular-unmuscular vascular border.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/308/308ra159/DC1

    Methods

    Fig. S1. Specific distal pulmonary arterioles muscularize with hypoxia.

    Fig. S2. Hypoxia exposure in mice induces KLF4 expression in PDGFR-β+ pulmonary arteriole SMCs.

    Fig. S3. Smooth muscle Klf4 deletion in SMA-CreER, Klf4(flox/flox) mice.

    Fig. S4. PDGFR-β+SMA+SMMHC+ cells are located at the new distally located muscular-unmuscular vascular transition zone at hypoxia day 21.

    Fig. S5. In human PA SMCs, KLF4 regulates hypoxia- and PDGF-BB–induced migration and hypoxia-induced migration.

    Fig. S6. Mosaic analysis of the role of KLF4 in distal pulmonary arteriole muscularization.

    Fig. S7. With hypoxic exposure, lung PDGF-B protein expression is enhanced and then polarized and finally down-regulated.

    Fig. S8. Lung ECs isolated from mice exposed to hypoxia have enhanced Pdgf-b expression.

    Table S1. Primer pair sequences for quantitative reverse transcription polymerase chain reaction.

    Reference (40)

  • Supplementary Material for:

    Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension

    Abdul Q. Sheikh, Ashish Misra, Ivan O. Rosas, Ralf H. Adams, Daniel M. Greif*

    *Corresponding author. E-mail: daniel.greif{at}yale.edu

    Published 7 October 2015, Sci. Transl. Med. 7, 308ra159 (2015)
    DOI: 10.1126/scitranslmed.aaa9712

    This PDF file includes:

    • Methods
    • Fig. S1. Specific distal pulmonary arterioles muscularize with hypoxia.
    • Fig. S2. Hypoxia exposure in mice induces KLF4 expression in PDGFR-β+ pulmonary arteriole SMCs.
    • Fig. S3. Smooth muscle Klf4 deletion in SMA-CreER, Klf4(flox/flox) mice.
    • Fig. S4. PDGFR-β+SMA+SMMHC+ cells are located at the new distally located muscular-unmuscular vascular transition zone at hypoxia day 21.
    • Fig. S5. In human PA SMCs, KLF4 regulates hypoxia- and PDGF-BB–induced migration and hypoxia-induced migration.
    • Fig. S6. Mosaic analysis of the role of KLF4 in distal pulmonary arteriole muscularization.
    • Fig. S7. With hypoxic exposure, lung PDGF-B protein expression is enhanced and then polarized and finally down-regulated.
    • Fig. S8. Lung ECs isolated from mice exposed to hypoxia have enhanced Pdgf-b expression.
    • Table S1. Primer pair sequences for quantitative reverse transcription polymerase chain reaction.
    • Reference (40)

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