**3. Discussion**

PAD is one of the common complications of DM that inflicts substantial damage to the lower limbs and has a very poor outcome. In mice, it was shown that impaired arteriogenesis is a major problem in hypercholesterolemia and DM [16]. While in the study by van Weel et al. [16], the influence of DM on perfusion recovery was less compared to that of hypercholesterolemia, our study showed a more pronounced impact of ligation on collateral vessel growth in DM. This may be due to the different experimental setup, age groups of animals under study, timepoints measured, and animal strains used. The mechanism by which impaired arteriogenesis occurs in DM is not fully understood. Earlier, Pagel et al. [22] reported the importance of *Egr-1* in promoting collateral outward remodeling through augmenting leukocyte infiltration and endothelial cell proliferation. In the present study, we report that there was a decreased expression of Egr-1 at the transcript and protein levels in collaterals of mice rendered diabetic by administration of streptozotocin. The decreased expression of *Egr-1* correlated with decreased collateral artery diameter in the diabetic mice. Important downstream targets of *Egr-1*, namely, *ICAM-1* and *uPA*, were found to be decreased, too. *ICAM-1* plays a critical role in endothelial monocyte adhesion, which is essential for arteriogenesis. An earlier study identified an upregulation of ICAM-1 mRNA in growing collateral arteries after induction of arteriogenesis by femoral artery ligation [25]. Moreover, it was shown that the process of arteriogenesis is reduced in ICAM-1 deficient mice [26]. Treatment of the diabetic mice with insulin improved the expression of *Egr-1*, increased the collateral artery diameter, and normalized the expression of downstream targets of *Egr-1*. A proposed model is shown in Figure 5.

**Figure 5.** Graphical representation showing the mechanisms by which Egr-1 controls leukocyte recruitment, and hence, the process of collateral artery growth. While this process is impaired in DM due to the reduced *Egr-1* expression, it can be rescued by insulin treatment.

Earlier studies have clearly shown that induction of arteriogenesis in C57Bl6 mice by FAL resulted in an increased expression of *Egr-1* at the transcript and the protein levels in growing collaterals [22]. Interestingly, *Egr-1* deficient mice have been shown to have increased basal levels of CD11b+ monocytes in the peripheral blood; however, levels of collateral perivascular macrophages as well as CD3+ T cells and CD19+ B cells in adductor muscles harvesting growing collaterals were reduced. Moreover, FAL in Egr-1−/− mice was associated with poor leukocyte recruitment and reduced collateral artery growth [22]. Our results showed that induction of DM, which was associated with reduced expression levels of *Egr-1*, also resulted in increased systemic levels of CD11b+ cells, and after induction of arteriogenesis, in reduced levels of CD11b<sup>+</sup>, CD3<sup>+</sup>, and CD19+ cells. Interestingly enough, treatment with insulin rescued perivascular leukocyte counts.

Decreased expression of *Egr-1* in DM mice in our study may support earlier findings showing that *Egr-1* is critical for collateral vessel development and that functional regulation of *Egr-1* may be compromised in DM. Evidence of induction and expression of *Egr-1* by elevated levels of glucose in murine glomerular endothelial cells and aortic smooth muscle cells have been reported. Exposure to insulin or high concentrations of D-glucose increased the expression of Egr-1 on the mRNA and protein level in glomerular endothelial cells and increased its promoter activity irrespectively of the concentration of insulin [27,28]. *TNF-*α is downstream of *Egr-1* and induces the expression of *MCP-1*. However, in arteriogenesis, increased levels of TNF-<sup>α</sup>, relevant for *MCP-1* expression, are dependent on mast cell activation [14]. Vedantham et. al. demonstrated a novel mechanism linking glucose metabolism to increased inflammatory and prothrombotic signaling in diabetic atherosclerosis via activation and post-translational modification of *Egr-1*. Hyperglycemia-induced hyper-acetylation of Egr-1 in endothelial cells was reported to be an important event linking diabetes to accelerated atherosclerosis [29]. Though acetylation of Egr-1 was not studied, it will be interesting to pursue future studies to understand the role of Egr-1 in the pathophysiology of arteriogenesis. Our observations in this study are contrary to those observed in diabetic atherosclerosis. There may be a possibility of additional regulation of Egr-1, as indicated by the shift in bands of Egr-1. Post-translational modifications of Egr-1 such as acetylation and phosphorylation have been reported to play an important role in the transcriptional activity and stability of Egr-1 [17,30]. Phosphorylation/dephosphorylation events may act as regulators for restricting the function of Egr-1. Furthermore, *SP-1* has been reported to compete for DNA binding sites of *Egr-1* [17]. Recently, a splice form of *Egr-1* was reported which lacks the N-terminal activation domain between amino acids 141 and 278 [31]. It will be interesting to further understand the mechanism through which hyperglycemia interferes with *Egr-1* upregulation during the process of arteriogenesis.

Leukocyte infiltration mediated through downstream target genes of *Egr-1*, namely, *ICAM-1, uPA* and *MCP-1*, was found to be decreased during collateral artery growth in diabetic mice. Endothelial

uPA is vital for neutrophil adherence to the endothelial cells [32]. Neutrophils accumulate around day 1 after FAL in the perivascular space of growing collaterals and have a relevant function in the recruitment of macrophages and lymphocytes, which appear at day 3 [14]. Indeed, it has been shown that *uPA* deficiency is associated with reduced perivascular leukocyte accumulation and results in reduced collateral artery growth after induction of arteriogenesis via FAL [33]. These data comply with reported findings that *Egr-1* mediates leukocyte infiltration through activation of the abovementioned genes in arteriogenesis [21]. Furthermore, the expression of the cell proliferation marker Ki-67 was found to be decreased in the growing collaterals of diabetic mice, highlighting the fact that, indeed, there was a decrease in the proliferation of vascular cells. SF-1, an important transcriptional repressor critical for smooth muscle proliferation and phenotype switch [33], was found to be elevated in our study in support of an earlier report demonstrating that Egr-1−/− mice exhibited increased expression of SF-1 in growing collateral arteries [21]. One of the interesting findings of our study as the beneficial effect of insulin on collateral artery growth in DM mice. SF-1 regulates gene expression of pro-inflammatory cytokines in smooth muscle cells [33] and antagonizes platelet-derived growth factor BB (PDGF-BB)-induced growth and differentiation of vascular cells [34]. Several earlier reports have shown a regulation of *Egr-1* by insulin. Furthermore, Gousseva et al. [28] have reported an insulin-mediated increase in *Egr-1* promoter activity and cell proliferation in bovine aortic smooth muscle cells. Egr-1 has a role in adipocyte insulin resistance through activation of the MAPK-ERK pathway [35]. Our studies clearly show that diabetic mice treated with insulin show an increased expression of *Egr-1*, accompanied by an augmented expression of *Egr-1* downstream target genes relevant for leukocyte recruitment. Indeed, our results show that insulin treatment, moreover, goes along with increased numbers of leukocytes—relevant for the process of arteriogenesis—in collateral harboring muscles.

In summary, *Egr-1* expression decreased after induction of arteriogenesis in growing collaterals of streptozotocin-induced diabetic mice compared to control mice. Decreased *Egr-1* expression led to poor collateral growth. Insulin treatment, however, normalized the *Egr-1* expression, thereby promoting arteriogenesis. Though this was an observational study, this study assumes significance as it is the first time associating *Egr-1* with impaired arteriogenesis in DM. It will be interesting to see whether the same processes occur in patients with DM. Further investigations exploring the mechanism by which hyperglycemia suppresses *Egr-1* expression during the process of arteriogenesis will lead to better understanding of impaired arteriogenesis in DM.

### **4. Materials and Methods**

### *4.1. Animal Studies*

All studies with mice were performed after approval by the Institutional and Local Animal Ethics Committee (CPCSEA Approval number: H01/SASTRA/IAEC/RPP-23/12/15). Male C57B6NTac (Taconic Biosciences, USA) mice were procured through Vivo Biotech Ltd., Telangana, India and were maintained in an air-conditioned room (25 ◦C) with a 12 h light/12 h dark cycle. Feed and water were provided ad libitum to all the animals. Mice were rendered diabetic by treating them with STZ according to published protocols [36]. Briefly, eight-week-old male mice were made diabetic by administration of 50 mg/kg STZ dissolved in fresh citrate buffer (0.05 mol/L, pH 4.5) i.p. per day for five consecutive days. Those mice displaying blood glucose levels ≥ 250 mg/dL were considered diabetic (DM mice). The non-diabetic (NDM mice) control mice received citrate buffer alone. One group of mice received insulin after confirmation of hyperglycemia. Insulin was administered subcutaneously at a daily dose of 0.20 mL/100 g (4–5 U) until the end of the experiment.

### *4.2. Femoral Artery Ligation and Collection of Collaterals*

Femoral artery ligation was performed as published earlier [37]. In brief, using a silk braided suture (0/7) the right femoral artery was ligated distally from the origin of the profunda femoris branching, while the left leg was sham operated. Adductor muscles and collateral arteries were collected as previously described [20]. To carry out fluorescent-activated cell sorting (FACS) analyses, adductor muscles were collected 3 days after the surgical procedure and for histological analysis, the adductor muscles were harvested 7 days after FAL. Gene expression studies on RNA and protein levels were performed with collateral arteries isolated 24 h after induction of arteriogenesis.

### *4.3. RNA Isolation and Quantitative Real-Time PCR Studies*

Gene expression studies were performed using quantitative real time PCR (qRT-PCR). Total RNA was isolated using the RNeasy kit (TaKaRA). After DNase I (Qiagen, Hilden, Germany) digestion, one microgram of total RNA was reverse transcribed by random hexamers and Superscript RT-PCR System (Invitrogen, Carlsbad, CA, USA). After purification, the cDNA was used for qRT-PCR using specific primers for Egr-1, MCP-1, ICAM-1, SF-1, and Ki-67 [21]. Results were normalized to the expression level of the 18S rRNA.

### *4.4. FACS Analyses of Blood and Muscle Tissue*

Whole blood withdrawn from the left ventricle 3 days post-ligation was analyzed by flow cytometry analyses (BD FACS Aria III, CA, USA) according to standard protocols. Furthermore, the adductor muscles from C57B6NTac mice were perfused with PBS (phosphate buffered saline) to eliminate the blood, harvested, and placed in small cell culture dishes. The tissue was cut into small pieces and digested 45 min at 37 ◦C using PBS buffer (50 mL) containing collagenase II (1 mg/mL), hyaluronidase (0.5 mg/mL) (both Sigma, St. Louis, MO, USA), dispase (1 mg/mL) (Gibco, Invitrogen, Carlsbad, CA, USA), and BSA (bovine serum albumin) (0.6 mg/mL) (Sigma). The suspension was then filtered with PBS/2%BSA through a 70 μm cell strainer (BD Falcon™), spun 10 min at 95 g, and the pellet finally resuspended in 100 μL PBS/2%BSA. The resulting cell suspension was analyzed by FACS using a panel of monoclonal antibodies against CD3 (T cells); (BioLegend Cat. No. 100201), CD11b (neutrophils, monocytes) (BioLegend Cat. No. 305902), CD19 (B cells) (BioLegend Cat. No. 115501), and CD45 (pan-leukocytes marker) (BioLegend Cat. No. 103101). Leukocyte populations were identified by fluorescence and scatter light characteristics. Cells from both peripheral blood and tissue were gated based on forward scatter (FSC-A)/side scatter (SSC-A). Leukocytes were identified by their positive staining with CD45. The final gating was based on CD45+/CD11b+, CD45+/CD19+, and CD45+/CD3+ cells (14).

### *4.5. Histological Analyses*

Histological analyses were performed on adductor muscles (harboring collateral arteries) isolated from C57B6NTac mice as described earlier [20]. Briefly, 7 days after the surgical procedure both hind limbs were perfused with PBS containing 0.1% adenosine and 0.05% BSA (Sigma), then 4 min with fixing solution (4% buffered paraformaldehyde) via cannulation of the aorta. Thereafter, tissue samples were paraffin-embedded, cut in cross-sections, and H&E staining was performed to measure luminal collateral artery diameters.

### *4.6. Western Blot*

Western blot analysis was performed on protein extracts, which were isolated from collaterals 24 h after femoral artery ligation or sham operation of DM and NDM mice according to standard procedures. Briefly, 30 μg of protein from all the tissue lysates was loaded onto a 10% sodium dodecyl sulfate (SDS) gel and ran at a power of 110 V. The protein in the gel was shifted to an immune-blot polyvinylidene difluoride (PVDF) membrane (1620112, Bio-Rad, USA) at 100 V for 1 h using Trans-Blot Turbo Transfer System (Bio-Rad). The blots were probed for Egr-1 protein (Egr-1 antibody (588)-Santa Cruz BioTechnology Cat.no #sc110). α-Tubulin served as a housekeeping protein (alpha tubulin antibody (B-7): Santa Cruz Biotechnology Cat.no. #sc-5286). The density of Egr-1 to α-tubulin was

measured through the Quant-One software (Bio-Rad). The immunoblots are a representation of at least three independent experiments.
