**3. Results**

#### *3.1. Erk1 KO Mice Exhibit Excessive but Poorly Functional Arteriogenesis*

To assess the role of ERK1 in arteriogenesis, we ligated the common femoral artery (CFA) of *Erk1*−/− mice and assessed blood flow recovery over time using laser Doppler imaging while the anatomical extent of arteriogenesis was studied using micro-CT. While blood flow recovery was reduced in *Erk1*−/− mice compared to wild type (WT) controls (Figure 1A,B), the micro-CT-determined extent of arteriogenesis was dramatically increased (Figure 1C,D). Since macrophages are the critical source of VEGF in this model and since the extent of arteriogenesis generally correlates with the amount of VEGF-A present [24], we used immunocytochemistry to assess the extent of macrophage accumulation in the arteriogenic zone. Staining with an F4/80 antibody of the thigh muscles around the area of CFA, ligation was carried out three and seven days following hindlimb ischemia. There was a marked increase in macrophage tissue infiltration in *Erk1*−/− mice compared to WT mice (Figure 1E). To confirm these findings, we carried out Western blotting using total thigh muscle lysates. In agreemen<sup>t</sup> with immunocytochemical findings, we observed a massive increase in F4/80 signal (Figure 1F).

**Figure 1.** *Erk1*−/− mice exhibit excessive but dysfunctional arteriogenesis. (**A**,**B**) Blood flow recoveryafter ligation of the CFA in *Erk1*−/− mice assessed by laser-Doppler right after the surgery and 3, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 10 mice) \* *p* < 0.05, \*\* *p* < 0.005using two-way ANOVA followed by Sidak's multiple comparison test. (**C**,**D**) Quantification of thehindlimb vasculature by micro-CT three weeks after femoral artery ligation. Bar graph represents meanwith SEM (*n* = 4 mice) \* *p* < 0.05, \*\* *p* < 0.005 using two-way ANOVA followed by Sidak's multiplecomparison test. (**E**,**F**) Macrophage infiltration in the thigh from *Erk1*−/− mice assessed by staining orWestern blotting. Scale bar, 50 μm. Bar graph represents mean with SEM (*n* = 3 mice) \* *p* < 0.05 using*t*-test.

7,

#### *3.2. Erk1 Deletions in Endothelial Cells, Macrophages, or Smooth Muscle Cells Do Not A*ff*ect Arteriogenesis*

To identify the cell type(s) involved in the defective arteriogenic phenotype seen in *Erk1*−/− mice, we created mice with two intronic loxP sites in the *Erk1* (*Mapk3*) gene (hereafter denominated as *Erk1Fl*/*Fl* (Figure 2A)). Since endothelial cells are critical to arteriogenesis [6], these mice were crossed with a strain carrying an inducible Cre recombinase under the control of the VE-cadherin promotor (Cdh5CreERT2) [25] generating a Cdh5CreERT2;Erk1f/<sup>f</sup> line (*Erk1iEC*−/−). Administration of tamoxifen to eight-week-old *Erk1Fl*/*Fl* mice resulted in a high e fficiency deletion of endothelial *ERK1* (Figure 2B). One week after tamoxifen treatment, common femoral arteries (CFA) of *Erk1iEC*−/− and control mice were ligated. Surprisingly, laser-Doppler assessment of blood flow recovery in *Erk1iEC*−/− mice showed that it was similar to that of WT control mice (Figure 2C,D). We next turned our attention to macrophages. Similarly to Cdh5 CreERT2, LysM Cre [26] was very e ffective in deleting *Erk1* (Figure 2E). Blood flow recovery after CFA ligation was not impaired compared to WT mice after either *Erk1* gene deletion (Figure 2F,G). We next deleted the *Erk1* gene in smooth muscle cells (SMC) using the Myh11CreERT2 driver line [27]. *Erk1* gene was e fficiently deleted in SMC (Figure 2H) and this deletion had no e ffect on the blood flow recovery after CFA (Figure 2I,J).

**Figure 2.** *Erk1* deletions in endothelial cells, macrophages, or smooth muscle cells do not affect arteriogenesis. (**A**) Generation of *Erk1* floxed mice by insertion of LoxP sites between exons 2 and 3 and exons 8 and 9. (**B**) Efficiency of *Erk1* deletion in endothelial cells was assessed by Q-PCR of endothelial cells isolated from mouse livers. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**C**,**D**) Blood flow recovery after ligation of the CFA in *Erk1*iEC−/− mice assessed by laser Doppler right after the surgery and 3, 7, and 14 days after the surgery. Bar graph represents mean with SEM (*n* = 8 mice). (**E**) Efficiency of *Erk1* deletion in macrophages was assessed by Q-PCR of peritoneal macrophages. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**F**,**G**) Blood flow recovery after ligation of the CFA in *Erk1*Mφ−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with

SEM (*n* = 6 mice). ( **H**) E fficiency of *Erk1* deletion in smooth muscle cells was assessed by Q-PCR of smooth muscle cells isolated from the mouse aortas. Bar graph represents mean with SEM (*n* = 4 mice) \* *p* < 0.05 using Mann–Whitney test. (**I**,**J**) Blood flow recovery after ligation of the CFA in *Erk1*SMC−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days after the surgery. Bar graph represents mean with SEM (*n* = 5 mice).

#### *3.3. Erk1 Deletions in Endothelial Cells and Macrophages Leads to an Excessive but Poorly Functional Arteriogenesis*

Finally, we bred mice with macrophage- (*Erk1* MφKO) and endothelial (*Erk1*iECKO)-specific deletions to generate double knockout mice with ERK1 expression disrupted in both cell types (*Erk1*iECKO/M<sup>φ</sup>KO). Induction of hindlimb ischemia in these mice led to impaired blood flow recovery that was similar to that observed in the *Erk1* global null mice (Figure 3A,B) and the anatomical extent of arteriogenesis was increased (Figure 3C,D). We carried out Western blotting using total thigh muscle lysates and we observed a massive increase in F4/80 signal (Figure 3E).

**Figure 3.** *Erk1* deletion in macrophages and endothelial cells leads to an excessive but poorly functional arteriogenesis. ( **A**,**B**) Blood flow recovery after ligation of the femoral artery in *Erk1*iEC−/−Mφ−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 6 mice) \* *p* < 0.05, \*\* *p* < 0.005 using two-way ANOVA followed by Sidak's multiple comparison test. ( **C**,**D**) Quantification of the hindlimb vasculature by micro-CT three weeks after femoral artery ligation. Bar graph represents mean with SEM (*n* = 6 mice) \* *p* < 0.05 using two-way ANOVA followed by Sidak's multiple comparison test. (**E**) Macrophage infiltration in the thigh from *Erk1*iEC−/−Mφ−/− KO mice assessed by Western blotting. Bar graph represents mean with SEM (*n* = 3 mice) \* *p* < 0.05 using *t*-test.

#### *3.4. Erk2 Deletions in Endothelial, but Not Other Cell Types, Decreases Arteriogenesis*

We next deleted endothelial *Erk2* (*Mapk1*) using the same Cdh5CreERT2 line. As in the case of *Erk1*, the administration of tamoxifen to eight-week-old *Erk2Fl*/*Fl* mice resulted in high efficiency deletion of the endothelial *Erk2* (Figure 4A). However, unlike the *Erk1iEC*−/− mice, the deletion of endothelial *Erk2* resulted in reduced flow recovery in *Erk2iEC*−/− compared to WT mice (Figure 4B,C). Surprisingly, there was no difference in the anatomical extent of arteriogenesis as determined by micro-CT imaging (Figure 4D,E). Endothelial nitric oxide synthase (eNOS) is a key enzyme producing the vasodilator NO and its activity is critical to arteriogenesis [28]. We found a decreased eNOS expression in *Erk2* KO endothelial cells compared to endothelial cells from WT mice (Figure 4F). We next focused on macrophages. LysM Cre was very effective in deleting *Erk2* in mice (Figure 4G). However, blood flow recovery after CFA ligation was not impaired compared to WT mice after *Erk2* gene deletion (Figure 4H,I). We next deleted the *Erk2* gene in smooth muscle cells using Myh11CreERT2 driver line (Figure 4J). *Erk2* deletion had no effect on the blood flow recovery (Figure 4K,L).

**Figure 4.** *Erk2* deletion in endothelial cells, but not in other cell types, decreases arteriogenesis. (**A**) Efficiency of *Erk2* deletion in endothelial cells was assessed by Q-PCR of endothelial cells isolated from mouse livers. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**B**,**C**) Blood flow recovery after ligation of the CFA in *Erk2*iEC−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 5 mice) \* *p* < 0.05 using two-way ANOVA followed by Sidak's multiple comparison test. (**D**,**E**) Quantification of the hindlimb vasculature by micro-CT three weeks after femoral artery ligation. Bar graph represents mean with SEM (*n* = 3 mice). (**F**) eNOS expression in endothelial cells isolated from livers from *Erk2*iEC−/− mice. \* *p* < 0.05 using Mann–Whitney test. (**G**) Efficiency of *Erk2* deletion in macrophages was assessed by Q-PCR on macrophages from the peritoneal cavity. Bar graph represents mean with

*Erk2* deletion in endothelial cells, but not in other cell types, decreases arteriogenesis. (**A**) Efficiency of *Erk2* deletion in endothelial cells was assessed by Q-PCR of endothelial cells isolated from mouse livers. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**B**,**C**) Blood flow recovery after ligation of the CFA in *Erk2*iEC−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 5 mice) \* *p* < 0.05 using two-way ANOVA followed by Sidak's multiple comparison test. (**D**,**E**) Quantification of the hindlimb vasculature by micro-CT three weeks after femoral artery ligation. Bar graph represents mean with SEM (*n* = 3 mice). (**F**) eNOS expression in endothelial cells isolated from livers from *Erk2*iEC−/− mice. \* *p* < 0.05 using Mann–Whitney test. (**G**) Efficiency of *Erk2* deletion in macrophages was assessed by Q-PCR on macrophages from the peritoneal cavity. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**H**,**I**) Blood flow recovery after ligation of the femoral artery in *Erk2*MΦ−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 4 mice). (**J**) Efficiency of *Erk2* deletion in smooth muscle cells was assessed by Q-PCR of smooth muscle cells isolated from mouse aortas. Bar graph represents mean with SEM (*n* = 4 mice) \*\* *p* < 0.005 using Mann–Whitney test. (**K**,**L**) Blood flow recovery after ligation of the femoral artery in *Erk2*SMC−/− mice assessed by laser-Doppler right after the surgery and 3, 7, and 14 days thereafter. Bar graph represents mean with SEM (*n* = 6 mice).

#### *3.5. ERK Isoform E*ff*ect on Endothelial Cell Proliferation and Migration*

To gain an insight into differences in ERK1- vs. ERk2-specific effects in the endothelium, we examined the effect of either isoform deletion on endothelial cell proliferation and migration. Administration of BrdU to P6 *Erk1*−/− mice showed no differences in the extent of endothelial cell proliferation in the retinal vasculature vs. WT controls (Figure 5A,B). In contrast, BrdU labeling in *Erk2iECKO* mice showed a ~50% reduction in endothelial proliferation. A combination of EC-specific *Erk2* and global *Erk1* knockouts (*Erk2iECKO*; *Erk1*−/− mice) did not result in a further decline in endothelial proliferation demonstrating that *Erk2* is the primary driver of this process. These findings were confirmed in vitro: *Erk2* but not *Erk1* knockdown resulted in decreased EC proliferation in an FGF2 growth assay (Figure 5C). In contrast, both ERKs were involved in EC migration response to VEGF-A or FGF2 stimulation in in vitro cell wounding assays (Figure 5D).

**Figure 5.** ERK isoform effect on endothelial cell proliferation and migration. (**A**,**B**) Assessment of endothelial cells proliferation in the retina of six-day-old pups by BrdU quantification. \* *p* < 0.05 compared to WT using Kruskel–Wallis test with Dunn's multiple comparison test. Scale bar, 50 μm. Bar graph represents mean with SEM (*n* = 5 mice). (**C**) Assessment of proliferation of pulmonary endothelial cells from WT mice (circle), *Erk1*−/− mice (square), or *Erk2*iECKO mice (triangle). White symbols are cells treated with vehicle, and black symbols cells treated with FGF2. Bar graph represents mean with SEM (*n* = 6 wells) \* *p* < 0.05, \*\* *p* < 0.005 compared to WT treated with vehicle using Kruskel–Wallis test with Dunn's multiple comparison test. (**D**) Assessment of migration of HUVEC treated with shRNA scrambled, or against *ERK1*, or against *ERK2* and stimulated with (VEGFA165 or FGF2. Bar graph represents mean with SEM (*n* = 4 wells) \* *p* < 0.05 compared to siSCR of each condition using Kruskel–Wallis test with Dunn's multiple comparison test.
