*2.1. Mice*

*Mapk3*−/− mice (denominated *Erk1*−/−), *Mapk1*tm1Gela/J mice (denominated *Erk2*Fl/Fl), and B6.129P2-Lyz2tm1(cre)Ifo/J mice (denominated LysMCre) were purchased from the Jackson Laboratory. *Cdh5CreERT2* mice were a generous gift from Ralf Adams. Myh11CreERT2 mice were a generous gift from Dan Greif. All mice, including the wild type (WT) mice, are *Mus musculus* on a pure C57Bl6 genetic background. *Erk1Fl*/*Fl* mice were realized by inserting 2 loxP sequences in introns between exons 2 and 3 and exons 8 and 9 of the Erk1 gene. Tamoxifen injections to induce deletion by the Cdh5Cre or Myc11Cre were done with 5 injections of 1.5 mg of tamoxifen on 5 consecutive days. Control mice received the same quantity of tamoxifen. For retinal angiogenesis, 100 μg of tamoxifen were administrated by IP injections starting at P1 to P4. BrdU was injected 2 h prior to euthanasia. Animals were housed and used in accordance with protocols and policies approved by the Yale Institutional Animal Care and Use Committee.

#### *2.2. Endothelial Cells, Macrophages, and Aortic Smooth Muscle Cell Isolations and Quantitative PCR*

Endothelial cells were isolated from mouse livers and lungs. Briefly, livers and lungs were collected and digested in a solution of collagenase and dispase (Roche/Sigma Aldrich, St Louis, MO, USA). The suspensions were then washed and filtered. Endothelial cells were isolated using magnetic beads anti-Rat IgG (Invitrogen, Camarillo, CA, USA) previously coated with rat anti-mouse CD31 antibody (BD). After extensive washing, cells were lysed and RNA was isolated using PicoPure RNa isolation kit (ThermoFisher, Waltham, MA, USA) or cultured.

Macrophages were isolated from the peritoneal cavity as previously described [23]. Macrophages were selected using magnetic beads anti-Rat IgG (Invitrogen) previously coated with rat anti-mouse F4/80 antibody (Invitrogen). After extensive washing, cells were lysed, and RNA was isolated using PicoPure RNa isolation kit (ThermoFisher).

Smooth muscle cells were isolated from the aorta. Aortas were collected and digested in 175 U/mL collagenase (Worthington), 1.25 U/mL elastase (Worthington, Lakewood, NJ, USA), and HBSS for 25 to 30 min at 37 ◦C. Adventitia layer was then pulled out. Media and endothelium were cut and

digested in 175 U/mL collagenase and 2.5 U/mL elastase in HBSS for 1 h at 37 ◦C. Endothelial cells were bound toon beads previously coated with rat anti-mouse CD31 antibody were used and discarded. The remaining smooth muscle cells were lysed and RNA was isolated using PicoPure RNa isolation kit (ThermoFisher).

cDNAs were synthetized with iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA) and qPCRs were performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad).

#### *2.3. shRNA Infection*

shRNA targeting ERK1 and ERK2 (Sigma-Aldrich, St Louis, MO, USA) were encapsulated into lentivirus that were then. Lentivirus were produced in 293T cells using second generation lentiviral system (Invitrogen).

#### *2.4. Hindlimb Ischemia Model*

This was done as previously described by our lab [6]. Laser Doppler flow-imaging was carried out using a Moor Infrared Laser Doppler Imager (LDI; Moor Instruments Ltd., Wilmington, DE, USA) under ketamine and xylazine anesthesia.

#### *2.5. Micro-CT Imaging*

Microcomputed tomography (micro-CT) of the hindlimb vasculature was done by injecting 0.7 mL bismuth contrast solution in the descending aorta and the vasculature was imaged and quantified as previously described [6].

#### *2.6. Western Blot*

Cells were lysed in RIPA buffer (Boston BioProducts, Ashland, MA, USA). Proteins were titrated using Bio-Rad Protein Assay Dye Reagent (Bio-Rad). A total of 20 ng of proteins were loaded on a 4–12% acrylamide gel (Bio-Rad) and then transferred on a PVDF membrane (Millipore). Primary antibodies used were: F4/80 (Invitrogen), ERK (Cell Signaling, Danvers, MA, USA), and β-actin (Sigma-Aldrich).

#### *2.7. Immunofluorescent Staining*

Frozen sections were treated with ice cold acetone. Permeabilization was done in triton 0.1%. Primary antibodies were: F4/80 (Invitrogen) and IsolectinB4 (Invitrogen). Pictures were taken using an SP5 confocal microscope (Leica, Allendale, NJ, USA).

#### *2.8. xCELLigence Real-Time Cell Analysis (RTCA)*

Endothelial cell proliferation was measured by using an xCELLigence RTCA instrument (Roche Diagnostics) and E-plate 16 (a modified 16-well plate, Roche Diagnostics). The E-plate 16 was coated with 0.1% gelatin, loaded with 100 μL cell-free medium, and left in a tissue culture hood for 30 min to reach equilibrium. The E-plate 16 was placed into the RTCA instrument to measure the background impedance. Thereafter, 100 μL cell suspensions with fewer than 3500 cells were added into each well of the E-plate 16, which was then placed in a tissue culture incubator for 30 min to allow cells to settle down before being measured by the RTCA device. The impedance value of E-plate 16 was automatically monitored every 15 min. FGF2 was added at 100 ng/mL.

#### *2.9. Endothelial Migration*

HUVEC migration was measured in a wound-healing assay, which used ibidi Culture-Inserts (ibidi) to generate the wound. An ibidi Culture-Insert has dimensions of 9 mm × 9 mm× 5 mm (width × length × height) and is composed of two wells. One or two inserts were placed into one well of a

six-well plate. After being coated with 0.1% gelatin, both wells of inserts were loaded with 100 μL cell suspension. FGF2 and VEGF were used at 100 ng/mL.

#### *2.10. Statistical Analyses*

Statistical tests were performed using the software GraphPad Prism 8.
