People living with lower extremity peripheral artery disease demonstrate vascular regenerative cell deficiencies: The PAD-VRCE CardioLink-17 study

Abstract

Objective

Vascular regenerative cell exhaustion (VRCE), a shift in the circulating progenitor cell profile, has been linked with adverse cardiometabolic outcomes. Whether VRCE is also involved in the pathogenesis and vascular repair mechanisms of peripheral artery disease (PAD) is unknown. In this study, we sought to compare the circulating vascular regenerative cell content of adults living with PAD with those without PAD.

Methods

PAD-VRCE CardioLink-17 (NCT06626646) was an observational cross-sectional study of 20 adults with symptomatic PAD and 20 without PAD. Circulating progenitor cell profiles were documented using a multiparametric flow cytometry assay based on the differential expression of aldehyde dehydrogenase activity in provascular progenitor cells combined with primitive and/or lineage-specific hematopoietic or endothelial cell markers.

Results

Individuals with PAD had a median (range) ankle brachial index of 0.82 (0.35-0.84). The two groups were well-matched for age and gender, but individuals with PAD had higher rates of dyslipidemia, higher weight, and blood pressure. Those with PAD had fewer circulating ALDH^hiSSC^lowCD133⁺ (44.0 ± 3.4% vs 49.7 ± 3.0%; P < .001) and ALDH^hiSSC^lowCD34⁺CD133⁺ (42.5 ± 4.9% vs 49.0 ± 4.9%; P < .001) cells, representing rare multipotent myeloid progenitors with vessel repair function. Individuals with PAD also had fewer ALDH^hiSSC^midCD86⁺CD163⁻ monocytes (2.5 ± 2.4% vs 4.1 ± 2.1%; P = .001), indicating a reduced monocytic contribution to systemic inflammation. By contrast, participants with PAD exhibited a higher frequency of ALDH^hiSSC^hi granulocyte precursors (0.48 ± 0.4% vs 0.22 ± 0.1%; P = .008), the primary drivers of systemic inflammation.

Conclusions

Our findings provide evidence of a novel association between PAD and the well-documented VRCE phenotype. Accordingly, reversal of VRCE may represent a potential therapeutic target in patients with PAD.

Clinical relevance

Vascular regenerative cell exhaustion may contribute to peripheral artery disease and may represent a novel and viable therapeutic opportunity for peripheral artery disease.

Article Highlights.

• •Type of Research: Multicenter, observational, cross-sectional study
• •Key Findings: Individuals with peripheral artery disease (PAD) (n = 20), vs. those without PAD (n = 20), had fewer circulating ALDH^hiSSC^lowCD133⁺ (44.0 ± 3.4% vs 49.7 ± 3.0%; P < .001) and ALDH^hiSSC^lowCD34⁺CD133⁺ (42.5 ± 4.9% vs 49.0 ± 4.9%; P < .001) multipotent myeloid progenitors with vessel repair function, fewer ALDH^hiSSC^midCD86⁺CD163⁻ proinflammatory monocytes (2.5 ± 2.4% vs 4.1 ± 2.1%; P = .001), and more proinflammatory ALDH^hiSSC^hi granulocyte precursors (0.48 ± 0.4% vs 0.22 ± 0.1%; P = .008).
• •Take Home Message: People with PAD have reduced frequencies of circulating ALDH^hiSSC^low CD34⁺/CD133⁺ cells.

Peripheral artery disease (PAD) is the third most common manifestation of atherosclerotic cardiovascular disease (ASCVD).¹ Estimates from the last decade indicate that >230 million people are living with PAD globally, and its prevalence is projected to increase, as low- and middle-income countries experience increased rates of noncommunicable disease alongside population aging.^1,2 Lower extremity PAD is characterized by systemic atherosclerosis of the lower limbs; patients with symptomatic lower extremity PAD usually present with intermittent claudication, which causes significant disability that can progress to rest pain and nonhealing ulcers in end-stage disease.³ The biological mechanisms underlying PAD are complex and multifactorial, although it is highly associated with type 2 diabetes mellitus (T2DM), obesity, and sedentary lifestyle.^4,5 Individuals with PAD are at an increased risk of a major adverse cardiovascular event (MACE).⁶

Peripheral ischemia due to PAD creates a hypoxic milieu that mobilizes hematopoietic cells (HPCs) and endothelial precursor cells (EPCs) from the bone marrow toward sites of vessel damage. Vascular repair mechanisms occur through three coordinated processes.⁷ First, angiogenesis is the expansion of vascular architecture through the formation of new capillary networks from pre-existing blood vessels, which is stimulated primarily by myeloid HPCs and circulating angiogenic monocytes.⁸ Second, vasculogenesis is the de novo synthesis of new blood vessels through the activity of exceedingly rare but proliferative endothelial colony forming cells (ECFCs), which generate the building blocks for new vessels.⁹ Finally, arteriogenesis, which is mediated by cells of the monocyte/macrophage lineage, involves the positive remodeling of pre-existing small collateral channels to form more functional vessels through extracellular remodeling.¹⁰ Accumulating evidence suggests that these homeostatic processes can be overwhelmed by vascular injury in inflammation-based cardiometabolic diseases such as T2DM, obesity, and ASCVD, compromising repair mechanisms and exacerbating the risk of future MACE.^6,11,12

The available evidence indicates that individuals with cardiovascular risk factors, including smoking history, dyslipidemia, and hypertension, live with a diminished vascular regenerative (VR) cell pool.13, 14, 15 Based on the well-established concept that high aldehyde dehydrogenase (ALDH) activity is higher in primitive HPCs and myeloid angiocrine cells (MACs), our group developed a novel high-throughput flow cytometry assay to quantify VR precursor cells in peripheral blood samples.^16,17 ALDH is a cytoprotective enzyme that protects long-lived progenitor cells from elevated oxidative stress. The expression of ALDH decreases up to 100-fold as precursor cells differentiate into more mature effector cell types. Through the quantification of circulating ALDH^hi cell content in peripheral blood using the ALDEFLUOR assay, we have demonstrated that primitive myeloid progenitor cells and circulating angiogenic monocytes possess previously established VR function.^16,18 Specifically, these cells are depleted in states of T2DM and obesity and among South Asian individuals with elevated cardiovascular risk profiles.19, 20, 21 Collectively, the findings indicate that this imbalance in VR cell content provides a quantitative measure of vascular regenerative cell exhaustion (VRCE), which is defined as the depletion and/or dysfunction of progenitor cell subsets with blood vessel repair capacity. Herein, we hypothesized that individuals living with PAD would demonstrate the VRCE phenotype compared with individuals without PAD. Accordingly, the aim of our study was to evaluate the relationship between PAD and the depletion of circulating VR progenitor cell content from both hematopoietic and endothelial lineages.

Cells originally described as EPCs in landmark literature have been subject to an evolution in classification from a broad cell population into distinct subgroups. Due to the inconsistent characterization of EPC populations with both hematopoietic and endothelial cell features, a consensus statement by Medina et al²² established EPCs as a heterogenous group of precursor cells comprising ultra-rare ECFCs and MACs. Importantly, MACs were restricted to the hematopoietic lineage, and they were 20-fold more abundant in blood than ECFCs established in culture.²³ We have shown that when isolated from fresh bone marrow or umbilical cord blood, the ALDH^hiSSC^low cell fraction co-expressing CD34⁺ and CD133⁺ represent a MAC rich population.^16,23 Medina et al²² also described the repair capacity of ALDH^low ECFCs after culture, as ALDH activity is gradually reduced during their expansion in culture.²⁴

Methods

Study design and participants

The PAD-VRCE (Vascular Regenerative Cell Exhaustion in adults with Peripheral Artery Disease; NCT06626646) was an observational cross-sectional study. Participants were identified from the medical records at three community clinics (study protocol Pro00080564 approved by the Advarra Institutional Review Board) and a tertiary care hospital (study protocol 24-124 approved by the Unity Health Toronto Research Ethics Board) in the Greater Toronto Area. Participants who were aged between 18 and 80 years were eligible if they did not fulfill any of the following exclusion criteria: cancer diagnosis within the last 5 years, presence of New York Heart Association Class IV heart failure, active liver or kidney disease, white blood cell count >15 × 10⁹/L, active antimicrobial treatment or steroid therapy, or presence of an autoimmune condition/acquired immunodeficiency. Participants were only entered into the study after they provided written informed consent, and they were classified into the PAD group (individuals with symptomatic lower extremity PAD and ABI < 0.85, a more stringent cut off than the diagnostic <0.9 to create a more homogenous cohort with a clear disease burden) or the non-PAD group (individuals with no evidence of PAD).

Reagents, antibodies, and commercial kits

The ALDEFLUOR kit (#01700) and assay buffer, ammonium chloride lysis buffer, and SepMate peripheral blood mononuclear cell isolation tubes (#85415) were obtained from STEMCELL. The kit and buffer were used as per manufacturer’s instructions. The CellRox Deep Red kit (#C10491; Thermo Fischer Scientific) was used to quantify the intracellular levels of reactive oxygen species (ROS). The following antibodies were sourced from BioLegend: CD49d-PE (no. 304304), CD86-PE-Dazzle-594 (no. 374218), CD45-PE-Cy7 (no. 982310), CD163-PE-Cy7 (no. 333614), EGFR-PE-Cy7 (no. 352910), CD34-APC (no. 378606), CD14-APC (no. 325608), CD66b-APC (no. 305118), CD133-BV-421 (no. 393908), and CCR3-BV-421 (no. 310714).

Isolation of peripheral blood mononuclear cells and analysis of progenitor cell content

Approximately 30 mL of peripheral blood was drawn from each participant. Progenitor cells were enumerated and characterized using a multiparametric flow cytometry assay that segregated cells according to ALDH expression and side scatter (SSC) properties.²⁵ ALDH expression enabled the segregation of ALDH^hi progenitor cells from more mature effector cells, and SSC (low, medium, or high) provided further definition based on intracellular complexity.

Mononuclear cells were isolated by centrifuging blood samples in SepMate tubes. In brief, red blood cells were lysed using an ammonium chloride solution (up to 3 times) and washed with phosphate buffered saline and 5% fetal bovine serum to eliminate cellular debris. The ALDEFLUOR assay was used to measure ALDH activity in peripheral blood mononuclear cells. Diethylamino benzaldehyde was used to inhibit low levels of ALDH activity and act as a negative control. Subsequently, samples were incubated with anti-human antibodies attached to fluorophores and analyzed through 6-color flow cytometry to identify progenitor cell subsets. Analysis was conducted on a CytoFLEX LX flow cytometer (Beckman Coulter) until 1 × 10⁶ cellular events were recorded. In instances where ALDH expression appeared as a gradient rather than a distinct population, unstained, diethylamino benzaldehyde, and fluorescence minus one controls were utilized to ensure consistent gating across samples shown by pseudocolor dot plots.

Study outcomes

The primary end point in the PAD-VRCE CardioLink-17 study was the difference in the frequency or absolute number of circulating ALDH^hiSSC^low primitive myeloid progenitor cells in individuals with PAD vs those without PAD. The secondary end points were the difference in the frequency or absolute number of circulating ALDH^hiSSC^mid monocytes and ALDH^hiSSC^hi granulocytes and the difference in ROS levels within ALDH^hi subsets between the two groups of participants.

Statistical analyses

Participant characteristics are reported as mean ± standard deviation for continuous variables or frequencies and percentages for categorical variables. Cellular frequencies are presented as mean ± standard error of the mean (SEM). Absolute cell counts are described as the mean total number of cells per 1 × 10⁶ events ± standard error of the mean. Between-group differences were assessed using two-sided Welch's t-test, which assumed an unequal variance for continuous variables. Categorical variables were analyzed using Fischer's exact test. All statistical analyses were performed using Prism 10 (GraphPad Software).

Results

Participant characteristics

The mean age of the full cohort was 60 ± 10.94 years, half of whom were male. In the PAD group, 16 individuals had intermittent claudication, and 4 had critical limb ischemia.

The average ABI for the PAD group was 0.82 (0.35-0.84). ABI was not recorded for the non-PAD group due to the lack of clinical suspicion, in accordance with Canadian guidelines.²⁶ Participants in the PAD group predominantly identified as South Asian (45%) or White (35%), and individuals in the non-PAD group were primarily South Asian (40%) or Southeast Asian (30%). Participants in the PAD group had a higher weight (P = .028; Table I). Individuals in the PAD group presented with higher mean systolic blood pressure (P = .024), although both groups had a comparable number of participants with a hypertension diagnosis. Compared with the non-PAD group, the PAD group had higher rates of dyslipidemia (P = .044), although lipid levels were comparable between the groups. Despite similar numbers of individuals with T2DM, more individuals in the PAD cohort were taking insulin (P = .047). The PAD group had a higher quantity of both white blood cells (P = .042) and total neutrophils (P = .006).

Table I.

Baseline characteristics of the PAD-VRCE CardioLink-17 participants

Variables	PAD	Non-PAD	P value
ABI	0.82 (0.35-0.84)	N/a
Age	61.3 ± 11.2	59.6 ± 10.6	.623
Male, %	10 (50)	11 (55)	>.999
Race
White	7 (35)	2 (10)	.127
South Asian	9 (45)	8 (40)	>.999
Black	3 (15)	1 (5)	.605
Southeast Asian	0 (0)	6 (30)	.020
Not specified	0 (0)	2 (10)	.487
Weight, kg	83.56 ± 18.61	71.06 ± 14.28	.028
BMI, kg/m2	30.74 ± 6.07	27.16 ± 5.49	.067
Blood pressure, mm/Hg
Systolic	138.85 ± 13.42	129.0 ± 11.96	.024
Diastolic	80.40 ± 10.29	79.36 ± 7.28	.713
Total cholesterol, mmol/L	4.25 ± 1.41	4.71 ± 1.05	.206
LDL-C, mmol/L	2.37 ± 0.99	2.77 ± 1.02	.225
HDL-C, mmol/L	1.23 ± 0.29	1.36 ± 0.35	.230
Triglycerides, mmol/L	1.54 ± 1.06	1.34 ± 0.55	.493
HbA1c, %	6.52 ± 1.16	5.99 ± 0.48	.086
eGFR, mL/min/1.73 m2	90 ± 17.93	97 ± 12.34	.129
Creatinine, μmol/L	74.79 ± 36.11	67 ± 11.68	.380
WBC, 109/L	7.62 ± 2.58	6.14 ± 1.60	.042
Neutrophils, 109/L	4.82 ± 1.81	3.3 ± 1.17	.006
Lymphocytes, 109/L	2.01 ± 0.69	1.99 ± 0.63	.916
Comorbidities
Type 2 diabetes mellitus	10 (50)	8 (40)	.750
Coronary artery disease	5 (25)	3 (15)	.695
Cerebrovascular disease	4 (20)	0 (0)	.106
Dyslipidemia	19 (95)	13 (65)	.045
Hypertension	15 (75)	10 (50)	.191
Smoking history	8 (40)	2 (10)	.065
Heart failure	3 (15)	0 (0)	.231
Medications
Metformin	7 (35)	2 (10)	.127
Insulin	5 (25)	0 (0)	.047
Statin	17 (85)	13 (65)	.273
SGLT2i	7 (35)	2 (10)	.127
GLP-1RA	6 (30)	2 (10)	.235
ACEi/ARB	12 (60)	6 (30)	.111
Calcium channel blocker	5 (25)	2 (10)	.408
Beta blocker	5 (25)	3 (15)	.695
Anticoagulants	4 (20)	1 (5)	.342

ABI, Ankle brachial index; ACEi/ARB, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker; BMI, body mass index; GLP-1RA, glucagon-like peptide one receptor agonist; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PAD, peripheral artery disease; SGLT2i, sodium-glucose co-transporter two inhibitor; WBC, white blood cell.

Data are presented as mean ± standard deviation or n (%). ABI presented as median (range).

ALDH^hi cell subpopulations

We utilized ALDH^hi activity in combination with increasing intracellular complexity to discern and enumerate MAC populations and exceedingly rare ECFC populations (ALDH^hiSSC^low), monocytes/macrophages (ALDH^hiSSC^mid), and granulocyte lineages (ALDH^hiSSC^hi).¹² Representative flow cytometry plots demonstrating the gating strategy for the analysis of ALDH^hi subpopulations is shown in Fig 1, A. ALDH^hiSSC^low cells represent a population of primitive HPCs and EPCs with previously described proangiogenic properties.^16,18,23 ALDH^hiSSC^mid cells are circulating monocytes capable of differentiating into either proinflammatory or anti-inflammatory polarized macrophages in tissues.²⁷ Although the ALDH^hiSSC^hi cell subpopulation represents granulocytic precursor cells involved in promoting inflammation, evidence suggests that the CD49d⁺ granulocyte subset may possess vessel repair function.²⁸ Although the frequencies of ALDH^hiSSC^low primitive progenitor cells and ALDH^hiSSC^mid monocytes in the blood samples were similar in this study, the frequency of ALDH^hiSSC^hi granulocytes was higher in the samples from the PAD group (P = .008; Fig 1, B-F). These distribution patterns were also found for absolute cell counts (Table II).

Fig 1.

Gating strategy and frequency of ALDH^hi subpopulations. A-C, Representative plots of gating for ALDH expression and ALDH^hi subpopulations utilizing pseudocolor mapping. D-F, Frequencies of ALDH^hi cells in participants with and without PAD (n = 20 for each group). ALDH, Aldehyde dehydrogenase; PAD, peripheral artery disease; SSC, side scatter.

Table II.

Frequencies and absolute counts of ALDH^hi cell subpopulations

Cell subpopulations	Cell frequencies			Absolute cell counts
	PAD	Non-PAD	P value	PAD	Non-PAD	P value
ALDHhiSSClow	0.037 ± 0.003	0.041 ± 0.005	.510	366.55 ± 33.21	411.87 ± 51.04	.473
CD34+	93.16 ± 0.56	93.23 ± 1.06	.955	343.70 ± 31.92	381.10 ± 51.16	.549
CD133+	44.03 ± 0.76	49.68 ± 0.67	<.001	166.32 ± 15.12	199.82 ± 24.63	.215
CD45+	79.23 ± 1.20	78.02 ± 1.70	.575	290.80 ± 27.15	314.46 ± 39.43	.633
CD34+CD133+	42.46 ± 2.05	48.99 ± 1.09	<.001	182.99 ± 20.98	158.08 ± 16.46	.377
CD34+CD133+CD45−	8.00 ± 0.63	10.06 ± 0.67	.038	37.58 ± 4.24	26.51 ± 2.05	.034
ALDHhiSSCmid	1.59 ± 0.26	1.45 ± 0.21	.666	15976 ± 2561	14474 ± 2122	.662
CD14+	97.95 ± 0.30	97.50 ± 0.38	.392	15658 ± 2535	14054 ± 2088	.637
CD86+	95.90 ± 0.51	96.46 ± 0.47	.440	15462 ± 2518	12556 ± 1565	.350
CD163+	95.88 ± 0.35	94.91 ± 0.65	.219	15374 ± 2519	13786 ± 2074	.638
CD14+CD86+	92.18 ± 0.89	89.69 ± 1.18	.109	13349 ± 1888	11617 ± 1423	.492
CD14+CD163+	94.38 ± 0.47	90.68 ± 1.18	.009	13365 ± 1853	13225 ± 1981	.961
CD86+CD163+	94.15 ± 0.71	91.42 ± 0.33	.002	15285 ± 2522	11887 ± 1501	.271
CD86+CD163−	4.11 ± 0.46	2.11 ± 0.24	.001	288 ± 32	525 ± 76	<.001
ALDHhiSSChi	0.48 ± 0.08	0.22 ± 0.03	.008	4754.51 ± 814.42	2188.59 ± 281.71	.010
CD66b+	88.62 ± 1.38	84.73 ± 2.19	.165	4246.93 ± 752.26	1848.62 ± 258.64	.009
CD49d+	14.47 ± 1.33	22.26 ± 2.66	.020	679.98 ± 97.21	409.32 ± 43.56	.022
CCR3+	2.27 ± 0.38	4.50 ± 0.47	.001	97.80 ± 13.02	121.61 ± 19.67	.336
EGFR+	1.17 ± 0.16	2.24 ± 0.40	.023	82.82 ± 18.76	44.49 ± 7.74	.077

ALDH, Aldehyde dehydrogenase; PAD, peripheral artery disease; SSC, side scatter.

Cell frequencies are presented as mean ± standard error of the mean. Absolute cell counts are presented as the mean number of cells per 1 × 10⁶ cellular events ± the standard error of the mean.

Diminished provascular progenitor cells in PAD

ALDH^hiSSC^low CD133⁺ cells are primitive progenitor cells with the capacity for hematopoietic reconstitution and with reported proangiogenic function in immunodeficient mice.^16,18 Our previous work demonstrated ALDH^hiSSC^low CD133⁺ cells as the most reliable population for the detection of reduced VR progenitor cell content.²⁰ In this study, we observed a significant reduction in the frequency of ALDH^hiSSC^low CD133⁺ cells in the PAD group compared with the non-PAD group (P < .001; Fig 2, A-C). Furthermore, our PAD cohort exhibited a diminished frequency of ALDH^hiSSC^low cells that co-expressed CD34 and CD133 (P < .001; Fig 2, D-F) despite having a similar frequency of CD34⁺ cells relative to the non-PAD group. We quantified the frequency of ALDH^hiSSC^lowCD45⁺ cells, revealing that approximately 80% of cells were of hematopoietic lineage in both groups. Individuals with PAD exhibited a reduction of CD34⁺CD133⁺CD45⁻ cells compared with those without PAD (P = .038). This subset of cells likely represents rare proliferative ECFCs that are generated in the bone marrow and mobilized into peripheral blood to participate in vasculogenesis.⁹ The significant decrease in proangiogenic progenitor cells represented by CD133 and CD34⁺CD133⁺ cells aligns with the findings of previous studies demonstrating VRCE in T2DM and obesity.^19,21 Cumulatively, these results indicate the severe depletion of MACs and ECFCs in individuals with PAD.

Fig 2.

Frequencies of ALDH^hiSSC^low CD133⁺ and CD34⁺CD133⁺ primitive progenitors in people with and without PAD. A and B, Representative flow plots with the gates for ALDH^hiSSC^low CD133⁺ cells. C, Frequencies of ALDH^hiSSC^lowCD133⁺ cells in participants with and without PAD (n = 20 for each group). D and E, Representative flow plots with the gates for ALDH^hiSSC^lowCD34⁺/CD133⁺ cells. F, Frequencies of ALDH^hiSSC^lowCD34⁺/CD133⁺ cells in participants with and without PAD (n = 20 for each group). PAD, peripheral artery disease; SSC, side scatter.

Effect of PAD on monocyte populations

To confirm that our ALDH^hiSSC^mid population was primarily composed of monocytes, we assessed CD14 expression, a pan-monocyte marker that was detected in >95% of ALDH^hiSSC^mid cells. Monocyte polarization toward a pro-/anti-inflammatory phenotype was determined through our assay based on CD86 and CD163 expression, respectively. Although no differences were noted in the frequency of ALDH^hiSSC^mid CD86⁺ or ALDH^hiSSC^mid CD163⁺ cells between the groups, we observed an increase in the frequency of ALDH^hiSSC^mid CD14⁺CD163⁺ monocytes in individuals with PAD (P = .01; Table II). The PAD group had a higher frequency of CD86⁺CD163⁺ cells than the non-PAD group (P = .002; Fig 3, A-C). The PAD group concurrently had a reduced frequency of committed proinflammatory macrophages denoted by CD86⁺CD163⁻ cells (P = .001; Fig 3, D) in comparison with the non-PAD group. These results suggest that monocyte polarization in participants with PAD was skewed toward an uncommitted inflammatory phenotype.

Fig 3.

Frequencies of macrophage precursor cells in people with and without PAD. A and B, Representative flow plots with the gates for ALDH^hiSSC^midCD86⁺/CD163⁺ and ALDH^hiSSC^midCD86⁺/CD163⁻ cells. C, Frequencies of ALDH^hiSSC^midCD86⁺/CD163⁺ cells in participants with and without PAD (n = 20 for each group). D, Frequencies of ALDH^hiSSC^midCD86⁺/CD163⁻ cells in participants with and without PAD (n = 20 for each group). PAD, peripheral artery disease.

People with PAD showed decreased granulocytes with vessel repair properties

An overall increase was observed in ALDH^hi granulocyte precursor cells in patients with PAD (P = .008). Subsequent characterization of ALDH^hiSSC^hiCD66b⁺ cells revealed no overall difference in the frequency of proinflammatory granulocytes between the groups.²⁹ Additional investigations of ALDH^hiSSC^hi cells were conducted for CD49d expression, a marker implicated in vessel integration and repair function after vessel damage by heat ablation.²⁸ Despite an overall increase in the frequency of granulocytic precursors, a pronounced decrease was found in the frequency of circulating ALDH^hiSSC^hi CD49d⁺ cells (P = .020; Fig 4, A-C) and chemokine receptor CCR3⁺ cells (P = .001; Fig 4, D-F) in individuals with PAD. Furthermore, a reduction was noted in the frequency of ALDH^hiSSC^hi EGFR⁺ cells (P = .023; Table II). Collectively, these differences indicate an increased systemic inflammatory burden in patients with PAD, accompanied by deficits in granulocytes associated with migration and vascular repair capacity.

Fig 4.

Frequencies of proangiogenic granulocytes in individuals with PAD. A and B, Representative flow plots with the gates for ALDH^hiSSC^hi CD49d⁺ cells. C, Frequencies of ALDH^hiSSC^hiCD49d⁺ cells in participants with and without PAD (n = 20 for each group). D and E, Representative flow plots with the gates for ALDH^hiSSC^hiCCR3⁺ cells. F, Frequencies of ALDH^hiSSC^hiCCR3⁺ cells in participants with and without PAD (n = 20 for each group). PAD, peripheral artery disease; SSC, side scatter.

People with PAD exhibited similar ROS levels

After live (SYTOX⁻) cells were isolated, ALDH^hi subpopulations were analyzed for ROS expression. The frequencies of ALDH^hiSSC^low ROX⁺ cells (P = .941), ADLH^hiSSC^mid ROX⁺ cells (P = .102), and ALDH^hiSSC^hi ROX⁺ cells (P = .375) were all equivalent in individuals with PAD and those without PAD.

Influence of smoking status and insulin therapy on circulating VR cells in PAD

As a secondary analysis, we investigated whether the well recognized CVD risk factors, smoking or insulin therapy for T2D, impacted circulating VR cells and in turn the primary outcome. However, no significant difference in circulating ALDH^hiSSC^low CD133⁺ cells was found in the PAD group stratified based on smoking history (smokers n = 8; nonsmokers n = 12; P = .724) or insulin dependence (insulin-dependent n = 5; noninsulin dependent n = 5; P = .557).

Discussion

The translational PAD-VRCE CardioLink-17 study provides the first evidence of a relationship between PAD and VRCE,²¹ that is characterized by lower frequencies of provascular progenitor cells and higher frequencies of proinflammatory monocytic and granulocytic precursors. As PAD is an inflammation-based disease, the data described herein align and expand upon our previous work that demonstrated decreases in circulating provascular progenitor cell content in proinflammatory milieu, including in the states of T2DM, obesity, and elevated lipoprotein(a) levels.^19,21,30 Previous work also established that individuals of South Asian descent with T2DM and/or ASCVD, an ethnic group disproportionately affected by T2DM and ASCVD, presented with similar deficiencies in provascular progenitor cell content compared with White Europeans.²⁰

Herein, we report a significant reduction in the frequency of circulating ALDH^hiSSC^low CD34⁺CD133⁺ cells, a cell population commonly associated with a proangiogenic secretome in HPCs, in the PAD group compared with the non-PAD group. Although the literature exploring this relationship is sparse, our results parallel that of Delva et al,³¹ who previously described a reduced quantity of CD133⁺ cells in individuals with PAD. Furthermore, Fadini et al³² demonstrated that concomitant PAD exacerbated the depletion of proangiogenic progenitor cells compared to individuals with T2DM alone.

Our results provide further evidence that provascular cell depletion can be demonstrated in the common manifestations of ASCVD. Vasa et al¹⁵ first described a 50% reduction in circulating MACs defined as CD34⁺KDR⁺ cells in individuals with coronary artery disease. Furthermore, an inverse association between cerebral ischemic events and the presence of CD34⁺ or CD133⁺ progenitor cells suggests that these cells are also implicated in the maintenance of cerebral vasculature and are associated with cerebrovascular disease.³³ Collectively, the results suggest that ASCVD is associated with a reduced frequency of circulating provascular progenitor cells, accompanied by an increase in risk of morbidity. Our results indicate that potential deficiencies in vascular repair in individuals with PAD may also extend to novel granulocyte precursor cells with associated vessel repair function. We observed that ALDH^hiSSC^hi CD49d⁺ cells were diminished in individuals with PAD. Cappellari et al²⁸ suggested that a deficiency of CD49d⁺ granulocytes was partially responsible for defective vessel repair in individuals living with type 2 diabetes. Additionally, we reported a reduction in the frequency of ALDH^hiSSC^hi CCR3⁺ granulocytes; these cells are similarly implicated in proangiogenic signaling.³⁴ Taken together, deficiencies in proangiogenic granulocyte content may be implicated in the reduced regenerative capacity in patients with PAD.

Macrophages derived from circulating monocytic precursors play a vital role in vascular repair.¹² Traditionally, ALDH^hiSSC^mid CD86⁺ cells are described as precursors to proinflammatory macrophages, and ALDH^hiSSC^mid CD163⁺ cells are considered to indicate the production of more anti-inflammatory macrophages.^35,36 Our study found that individuals with PAD had a reduced frequency of committed proinflammatory ALDH^hiSSC^mid CD86⁺CD163⁻ monocytes, an observation contrary to our laboratory previous work in individuals with diabetes and obesity.^19,21 Given the pathological differences between PAD and the more metabolic-based T2DM and obesity, the differential VR profiles observed may be partially attributed to an ischemia-driven need for arteriogenesis in PAD.

The reduced frequency of provascular progenitor cells and proangiogenic granulocytes combined with the alterations observed in monocyte precursor populations suggest multifactorial impairment in VR capacity in patients with PAD. The mechanisms implicated in VR cell depletion include elevated oxidative stress, chronic inflammation, dysregulation of angiogenic pathways, and endothelial dysfunction; these mechanisms are common across cardiovascular disease states. The exact drivers of VRCE may differ between PAD and metabolic diseases such as T2DM, but the cellular phenotype produced by these states appears to be consistent. This shared deficit in circulating VR cell content may be associated with common pathways through which diverse cardiovascular risk factors compromise vascular repair.

Werner et al³⁷ first demonstrated that individuals with coronary artery disease with decreased EPCs, which were subsequently reclassified as MACs, were more likely to develop MACE. This seminal contribution first correlated reduced MAC content combined with increased endothelial dysfunction as a mechanism underlying worsening outcomes. Therefore, individuals with PAD who have these deficits may be at an increased risk of MACE.³⁸

The results of this study must be viewed in the context of its limitations. First, the sample size of 40 was small, and the PAD group primarily consisted of individuals with claudication symptoms, limiting the overall generalizability of our results to individuals further along the PAD spectrum. Furthermore, with this sample size, the subanalyses of the influence of key patient characteristics were likely underpowered. Second, the cross-sectional design prevented the temporal assessment of VR cell profiles. Lastly, the functional effect of PAD was not evaluated in this study, although investigations into this topic are ongoing.

Pharmacologic (empagliflozin, icosapent ethyl, and semaglutide) and surgical strategies have been reported to improve circulating VR progenitor cell content in proinflammatory states.^19,39, 40, 41 Notably, semaglutide, which increased the pain-free walking distance of people with PAD and intermittent claudication in patients from the Semaglutide and walking capacity in people with symptomatic PAD and type 2 diabetes (STRIDE) trial, has also been found to reverse VR cell depletion in individuals with T2DM.^41,42 Taken together, the combined translational and clinical trial evidence confirms that VR cells are a potential therapeutic target for PAD.

Conclusions

To the best of our knowledge, these findings provide the first evidence of potential vascular repair deficits with a proinflammatory microenvironment in people with lower extremity PAD. However, future investigations with a larger sample size and prospective follow-up are necessary to establish a causal pathological relationship between PAD and circulating progenitor cell frequencies. Conducting a more extensive trial to include functional end points of VR cell subsets will provide further insights into the relationship between VRCE and clinical outcomes, facilitating future novel therapeutic advances that improve the lives of individuals with PAD.

Author Contributions

Conception and design: JH, MM, HT, GR-N, GC, DH, SV

Analysis and interpretation: JH, MM, HT, AK, AQ, YP, CM, CDM, MH, DH, SV

Data collection: JH, MM, AK, YP, FD, AH, BP, RV, EM, SH, CJD, GM, CDM

Writing the article: JH, HT, AK, DH, SV

Critical revision of the article: JH, MM, HT, AK, AQ, YP, FD, AH, BP, RV, EM, SH, CD, GM, CM, CDM, MH, GR-N, GC, DH, SV

Final approval of the article: JH, MM, HT, AK, AQ, YP, FD, AH, BP, RV, EM, SH, CD, GM, CM, CDM, MH, GR-N, GC, DH, SV

Statistical analysis: JH, DH, SV

Obtained funding: DH, SV

Overall responsibility: JH, MM, HT, AK, AQ, YP, FD, AH, BP, RV, EM, SH, CD, GM, CM, CDM, MH, GR-N, GC, DH, SV

Funding

None.

Disclosure

H.T. reports personal fees from the Canadian Medical and Surgical Knowledge Translation Research Group and LMC Healthcare. G.M. reports personal fees from the Canadian Medical and Surgical Knowledge Translation Research Group. C.D.M. is supported by a Merit Award from the University of Toronto Department of Anesthesiology and Pain Medicine, holds the Cara Phelan Chair in Critical Care at St Michael's Hospital-Unity Health Toronto, and has received advisory board honoraria/consulting fees from Amgen, Alexion, AstraZeneca, BioAge, Biotest, Boehringer Ingelheim, Cardior, Cytosorbents, Delex, ONA, PhaseBio, Sandoz, Trimedic Therapeutics, VarmX, and Werfen, as well as data safety monitoring board stipends from Beth Israel Deaconess Medical Center, Cerus, and Takeda. M.A.H. reports research grants from Vascular Therapies (ACCESS-2 Trial), Humacyte (V-012 Trial), VenoStent (SAVE-FistulaS Trial), Voyager Biomedical (ACT II Trial), and Laminate Technologies (Post Market Study for VasQ) and consulting fees from Humacyte and Venova. D.A.H. holds the Sheldon H. Weinstein Endowed Chair in Diabetes Research at Western University. S.V. holds a Tier 1 Canada Research Chair in Cardiovascular Surgery and reports receiving grants and/or research support and/or speaking honoraria from Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Canadian Heart Research Centre, Canadian Medical and Surgical Knowledge Translation Research Group, Eli Lilly, HLS Therapeutics, Humber River Health, Janssen, Merck, Novartis, Novo Nordisk, Pfizer, PhaseBio, S & L Solutions Event Management Inc, Sanofi, and Sun Pharma. The other authors have no relevant conflicts of interest to declare.

From the New England Society for Vascular Surgery