Fig 2: Contribution of myocyte-specific hypoxia-inducible factor (HIF) isoforms Hif1a or Hif2a to cardioprotection. a Schematic of breeding approach to generate mice with induced myocyte-specific HIF deletions used in subsequent studies. Hif1aloxP/loxP or Hif2aloxP/loxP mice were crossed with Cre-recombinase expressing mice under the control of Myosin-heavy chain promoter (Myosin-Cre+); these mice express cre-recombinase under the control of a tamoxifen-inducer. Control animals (Myosin-Cre+), Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ received a daily dose of 1 mg i.p. Tamoxifen five consecutive days to induce the Cre-recombinase activity. After 7 days, animals underwent experimental protocol (60 min of in situ myocardial ischemia followed by 120 min of reperfusion). b HIF1-alpha or HIF2-alpha immunoblot analysis from homogenized myocardial tissue, harvested from male Myosin-Cre+ and Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ mice, matched in age and weight. Mice underwent a thoracotomy with no further treatment(-I) or 60 min of myocardial ischemia (+I) followed by 120 min reperfusion; ß-Actin (ACTb) served as a loading control. One representative blot out of three experiments is shown. c, d Quantification by densitometry of the HIF-immunoblot results relative to ACTb. Data are expressed as mean fold change ±SD normalized to untreated myocardial tissue from Myosin-Cre+ compared by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 3 per group; c: F5,12 = 6.74, p = 0.0033; d: F5,12 = 21.85, p < 0.0001). e Infarct sizes ±SD in Myosin-Cre+, Hif1aloxP/loxP Myosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ mice, presented as the percentage to the area-at-risk after 60 min of ischemia, followed by 120 min of reperfusion (Myosin-Cre+ n = 5; Hif1aloxP/loxPMyosin-Cre+ n = 5; Hif2aloxP/loxPMyosin-Cre+ n = 4; per group mean ± SD; compared by one-way ANOVA followed by Bonferroni’s multiple comparison test; F2,11 = 7.901; p = 0.0075). f Representative infarct staining from Myosin-Cre+, Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+. g Troponin serum levels after 60 min ischemia, followed by 120 min of reperfusion in Myosin-Cre+, Hif1aloxP/loxPMyosin-Cre+ or Hif2aloxP/loxPMyosin-Cre+ (Myosin-Cre+ n = 5; Hif1aloxP/loxPMyosin-Cre+ n = 5, and Hif2aloxP/loxPMyosin-Cre+ n = 4 per group; presented as mean ± SD; compared by one-way ANOVA followed by Bonferroni’s multiple comparison test; F2,11 = 19.14, p = 0.0003)

Fig 3: Divergent changes in HIF1a protein levels induced by antioxidant enzymes capable of scavenging peroxide cannot be attributed to differential changes in Hif1a mRNA synthesis or stability.(A, B) Relative changes in Hif1a mRNA in SH-SY5Y cells in response to forced expression of various antioxidant enzymes. Data were pooled from three independent experiments in the form of mean ± SD. One-way ANOVA with Dunnett’s post-test was used for comparing cells expressing catalase or GPX1 with respect to cells expressing GFP and Student’s t test was used for comparing cells expressing PRDX3 with respect to GFP. (n.s.) indicates non-significant difference with respect to respective GFP controls under normoxia while (ns) and (#) indicate non-significant difference and the statistical difference of p < 0.05, respectively, with respect to GFP control in hypoxia. (C, D, E) Changes in half-life of HIF1a in SH-SY5Y ODD-Luc cells expressing catalase or GPX1 or PRDX3 with respect to that of respective GFP controls in hypoxia. ODD Half-life was assessed by performing a pulse chase experiment by adding 35 µM cycloheximide at every 20 min for a total of 4 hr using luciferase activity assay in SH-SY5Y cells expressing these antioxidants pre-treated either with or without 10 µM MG132. Data were pooled from three independent experiments in the form of mean ± SD. Two-way ANOVA with Bonferroni’s post-test was used for statistical analysis. (*), (**), and (***) indicate statistical differences of p < 0.05, p < 0.01, and p < 0.001 with respect to respective GFP controls in hypoxia. (°°°), (???) and (#) represent statistical differences of p < 0.001w.r.t CAT, p < 0.001 w.r.t GPX1 and p < 0.05 w.r.t PRDX3, respectively.

Fig 4: HIF-1a loss is observed in OA cartilage from human and mice.(A) Immunofluorescence staining of HIF-1a in articular cartilage of Lcp1-/- mice and WT littermates at 4 and 8 weeks after ACLT. Scale bar, 20 µm. (B) Quantitative analysis of HIF-1a fluorescence intensity in Lcp1-/- and WT mice articular cartilage. (C) Safranin O/fast green staining of human tibia articular cartilage in different OARSI grades. Scale bar, 400 µm. (D) Immunofluorescence staining of HIF-1a in human tibia articular cartilage with different OARSI grades. Scale bar, 100 µm. (E) Quantitative analysis of HIF-1a fluorescence intensity in human articular cartilage with different OARSI grades. (F and G) Violin plots showing the GSVA score of HIF1 and OA signaling pathway in five major cell types. n = 5 per group. **P < 0.01 and ***P < 0.001. FC, fibrocartilage chondrocyte; preHTC, prehypertrophic chondrocyte; rHTC, regulatory hypertrophic chondrocyte; OA-HTC, OA-associated hypertrophic chondrocyte; ProHTC, proliferative chondrocyte.

Fig 5: (a) Photos of HT1080 sarcoma xenografts treated with control IgG or DC101 and stained for hypoxia using Hypoxyprobe™-1. Length of treatment (Tx) and tumor sizes at time of harvesting are listed. (b) Percent hypoxia of H1080 xenografts at various sized tumors after treatment with control IgG or DC101. (c) Percentage of nuclei in various sized tumors treated with control IgG or DC101 staining for HIF-1a or HIF-2a. Bars represent standard deviation. *p < 0.05 compared to control IgG group; ns, not significant (p > 0.05). (d) Photos of HT1080 sarcoma xenografts treated with control IgG or DC101 and stained for hypoxia HIF-1a. Tumor sizes at time of harvesting are listed. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]