Preserved function of late outgrowth endothelial cells in medically-treated hypertensive patients under well-controlled conditions

Abstract

Endothelial-progenitor-cells participate in renal repair, but their number and function may be impaired by exposure to cardiovascular risk factors. The number of circulating endothelial-progenitor-cells is decreased in essential and renovascular hypertensive patients, but the effects of hypertension on endothelial-progenitor-cell function are incompletely understood. We hypothesized that endothelial-progenitor-cell function was preserved under well-controlled conditions in treated hypertensive patients. Patients with atherosclerotic-renal-artery-stenosis (n=22) or essential-hypertension (n=24) were studied during controlled sodium intake and anti-hypertensive regimen. Late-outgrowth-endothelial-progenitor-cells were isolated from the inferior vena cava and renal vein blood of atherosclerotic-renal-artery-stenosis and essential-hypertension patients, and a peripheral vein of matched normotensive controls (n=18). The angiogenic function of endothelial-progenitor-cells was assessed in vitro and multi-detector computer tomography used to measure single-kidney hemodynamics and function in atherosclerotic-renal-artery-stenosis and essential-hypertension patients. Inflammatory biomarkers and endothelial-progenitor-cell homing signals levels and renal release were calculated. Inferior vena cava and renal vein-obtained endothelial-progenitor-cell function were similar in atherosclerotic-renal-artery-stenosis and essential-hypertension patients, and comparable to that in normal controls (tube length 171.86±16.846, 191.09±14.222, 174.925±19.774μm, respectively). Function of renal vein-obtained endothelial-progenitor-cells directly correlated with stenotic-kidney glomerular filtration rate, endothelial-progenitor-cell homing factors and anti-inflammatory mediator levels in atherosclerotic-renal-artery-stenosis patients. Therefore, endothelial-progenitor-cell function was relatively preserved in atherosclerotic-renal-artery-stenosis patients, although it directly correlated with renal function. Adequate endothelial-progenitor-cell function supports the feasibility of using autologous endothelial-progenitor-cells as a therapeutic option in essential and renovascular hypertensive patients. Homing signals and inflammatory mediators may potentially regulate endothelial-progenitor-cell angiogenic function.

Introduction

Atherosclerotic renal artery stenosis (ARAS) is the major cause for secondary hypertension, which is characterized by reduced renal perfusion. Population studies revealed that ARAS constitutes an important risk factor for cardiovascular morbidity and mortality compared with essential hypertension (EH)¹. We have also previously shown that ARAS exacerbates renal inflammation, oxidative stress, and renal release of cytokines in both experimental models² and patients³.

A large body of evidence has shown that circulating bone-marrow derived endothelial progenitor cells (EPCs)-contribute to endothelial repair in ischemic tissues⁴. These cells have been described to include two subpopulations, based on the stage at which they appear in culture. Early-outgrowth-endothelial-cells (EOEC) appear after 4–7 days, and show monocytic features, and late-outgrowth-endothelial-cells (LOEC) appear after 14–21 days in culture, possess salient angiogenic capabilities, and exhibit many features resembling bone marrow-derived circulating EPC⁵. Furthermore, a combination of autologous EOEC and LOEC expanded in vitro has been used to replenish injured kidneys and improve their function in chronic experimental ARAS ^6–8.

However, EPCs function may be impaired by exposure to cardiovascular risk factors like hypertension or diabetes^{9, 10}, partly due to decreased nitric oxide availability or activation of the renal-angiotensin-aldosterone system (RAAS). Activation of angiotensin-II (Ang-II) in ARAS is involved in atherogenesis, inflammation, oxidative stress and endothelial damage¹¹, and is particularly prominent within the kidney. Furthermore, we have previously shown that the number of systemic circulating CD34⁺/KDR⁺ EPC is decreased in both ARAS and EH patients compared to healthy volunteers (HV), yet is further decreased in the renal rein (RV) effluent of the post-stenotic ARAS kidneys (STK) compared to EH kidneys. Pertinently, we have demonstrated that inflammatory cytokines released from the human STK recruited and sequestered circulating EPCs to participate in the reparative process³. Nevertheless, EPC crossing the circulation of the STK might be functionally damaged because of the noxious microenvironment. Importantly, impaired LOEC function would imply impaired endogenous renal regeneration capacity in ARAS patients, and argue against use of LOEC to replenish the diminished population of EPCs available for renal repair.

However, it remains unknown whether LOEC function in ARAS patients is adequate and affords reparative activity. The current study was designed to test the hypothesis that angiogenic function of LOEC expanded from the systemic circulation and STK of ARAS patients is preserved compared to that in EH and HV.

Methods

This study was approved by the Institutional Review Board of the Mayo Clinic. ARAS (n=22) or EH (n=24) patients were prospectively enrolled, and HV (n=18) without cardiovascular risk factors prospectively recruited through the Mayo Clinic Biobank.

In all EH or ARAS patients, the antihypertensive regimen included RAAS blockade. During a 3-day inpatient protocol, STK renal blood flow (RBF) and glomerular filtration rate (GFR) were measured using multi-detector CT (MDCT). Blood samples were collected from the inferior vena cava (IVC) and renal vein (RV) of ARAS and EH patients and from peripheral vein of HV for measurements of inflammatory markers levels, EPC isolation, and subsequent evaluation of their proliferation, migration, and tube formation function in-vitro. For detailed methodologies please see the online Data Supplement.

Results

Patients Characteristics

The characteristics of the study subjects are summarized in Table 1. Systolic blood pressure was higher in EH and ARAS patients compared with HV. Antihypertensive medications were similar between EH and ARAS.

Table 1.

Characteristics of healthy volunteers, essential hypertensive and atherosclerotic renal artery stenosis patients

Characteristics	HV(n=18)	EH(n=24)	ARAS(n=22)	P value
Age (years)	69.1±1.8	69.8±1.5	70.2±1.5	0.88
Gender (male/female)	11/7	15/9	12/10	0.85†
BMI (kg/m2)	26.3±1.0	27.0±0.9	27.8±0.9	0.54
Degree of stenosis (%)	0.0±0.0	0.0±0.0	74.2±0.7	<0.0001
Systolic pressure (mmHg)	120.4±2.8	132.5±2.4*	137.8±2.6*	<0.0001
Diastolic pressure (mmHg)	70.8±1.9	69.3±2.3	69.9±2.8	0.57
MAP (mmHg)	87.7±1.7	90.3±1.9	90.9±2.1	0.50
Total cholesterol (mg/dl)	177.7±6.7	174.8±5.8	170.8±6.0	0.76
Triglycerides (mg/dl)	117.1±17.5	123.8±11.4	133.7±13.3	0.26
HDL (mg/dl)	57.0±3.6	53.1±2.7	55.1±4.4	0.42
LDL (mg/dl)	90.3±4.6	96.8±4.4	88.9±4.7	0.43
Serum creatinine (mg/dl)	0.93±0.04	1.03±0.06	1.24±0.07*	0.005
Proteinuria (mg/24h)	86.9±7.4	90.5±13.2	76.6±11.0	0.24
Microalbuminuria (mg/24h)	0/0	33.0±5.9	29.1±8.1	0.68
Systemic PRA (ng/ml/h)	0.51±0.1	6.4±1.4*	8.9±1.8*	0.0002
Calcium-channel blocker	0/0	9/37.5	8/36.4	0.84†
ACE-I	0/0	16/66.7	12/54.5	0.40†
Angiotensin receptor blockers	0/0	12/50.0	15/68.2	0.21†
Alpha-blocker	0/0	2/8.3	1/4.5	0.60†
Statins	0/0	13/54.2	14/63.6	0.51†

Data are mean±SEM or (n, %). HV: healthy volunteers; EH: essential hypertension; ARAS: atherosclerotic renal artery stenosis patients; BMI: body-mass index; MAP: mean arterial pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein, PRA: plasma renin activity; ACE-I: angiotensin-converting enzyme inhibitor.

^*p<0.05 vs HV.

^†fisher’s exact test, between EH and ARAS.

Kidney function

Serum creatinine levels were elevated in ARAS compared to HV (Table 1), but urine protein excretion was unaltered. Systemic PRA levels were elevated in both ARAS and EH compared with HV.

Single-kidney function in the three groups is presented in Table S1. Of the ARAS patients, 10/22 were found to have bilateral disease, in which case both kidneys were considered as STK. Single-kidney cortical perfusion was decreased in STK compared with both EH and the non-stenotic, contralateral kidneys (CLK), and in the CLK compared with EH. Medullary perfusion was decreased in the STK compared to EH, while STK-RBF and GFR were both reduced compared to EH and CLK. RV PRA was elevated in the STK compared with EH.

LOEC function in vitro

Cultured LOEC had a cobblestone appearance (indicative of endothelial-like phenotype) and expressed CD34 and KDR (Figure S1 A–B). Migration activity, proliferation, and tube formation of LOEC obtained from either collection site was all similar among the three groups (Figure 1A–F).

Figure 1.

Extra-renal (peripheral or inferior vena cava) and renal vein levels of late outgrowth endothelial cell (LOEC) migration, proliferation and tube formation capacity in healthy controls, essential hypertensive (EH) patients, or the stenotic kidneys (STK) and contralateral kidneys (CLK) of patients with atherosclerotic renal artery stenosis (ARAS). LOEC function was similar among the groups regardless of whether they were expanded from the extra-renal (A–C) or renal (D–F) veins. Bottom panel: Representative tube formation images.

The STK and CLK-RV-LOEC migration and proliferation, respectively, directly correlated with the corresponding single-kidney GFR in ARAS, and their migration with CLK cortical perfusion. The STK-RV-LOEC migration tended to correlate with STK cortical perfusion (Figure 2A–D). The presence of bilateral disease did not significantly affect these relationships (Figure S2).

Figure 2.

Correlation of late-outgrowth-endothelial-cell (LOEC) function with renal hemodynamics, glomerular filtration rete (GFR), inflammatory markers or homing signals in healthy volunteers (HV), atherosclerotic-renal-artery-stenosis (ARAS) and essential hypertensive (EH) patients. (A–D) In ARAS, renal vein (RV)-LOEC migration and proliferation directly correlated with single-kidney GFR in stenotic (STK) and contralateral (CLK) kidneys, and migration with CLK cortical perfusion. The STK-RV- LOEC migration tended to correlate with STK cortical perfusion. (E–F) Conversely, EH-RV-LOEC migration inversely correlated with single-kidney cortical perfusion and GFR. (G–I) The EH-RV-LOEC proliferation correlated inversely with systolic blood pressure (SBP), and tube formation with diastolic blood pressure (DBP) and mean arterial pressure (MAP). (J–K) Tube formation of CLK-RV and HV systemic LOEC directly correlated with the level of granulocyte-macrophage colony-stimulating factor (GM-CSF). (L–M) Similarly, both systemic and RV-LOEC migration respective directly correlated with the level of GM-CSF in EH patients. (N–R) In ARAS, the STK-RV-LOEC migration directly correlated with the renal release of granulocyte CSF (G-CSF) and interleukin (IL)-10. Also, the systemic-LOEC migration directly correlated with the of stromal-derived factor (SDF)-1 level, and RV-LOEC migration with monocyte chemoattractant protein (MCP)-1. (R–S) IVC-LOEC migration inversely correlated tumor necrosis factor (TNF)-α level in ARAS and tube formation of EH RV-LOEC with interferon (IFN)-γ.

Conversely, the EH-RV-LOEC migration correlated inversely with the corresponding cortical perfusion and GFR (Figure 2E–F). In addition, EH-RV-LOEC proliferation inversely correlated with systolic blood pressure, and tube formation with diastolic and mean arterial pressure (Figure 2G–I).

We have also observed significant correlations between LOEC functions and homing and inflammatory mediator levels in the corresponding sites. The HV and CLK-RV-LOEC tube formation directly correlated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (Figure 2J–K). Similarly, both systemic and RV-LOEC migration directly correlated with IVC and RV level of GM-CSF in EH, respectively (Figure 2L–M), and the STK-RV-LOEC migration directly correlated with the renal release of granulocyte-CSF (G-CSF) and interlukin (IL)-10 (Figure 2N, R). The ARAS-IVC-LOEC migration also directly correlated with stromal-derived factor (SDF)-1, and that of STK and CLK-RV with monocyte chemoattractant protein (MCP)-1 (Figure 2O–Q). Moreover, the ARAS-systemic-LOEC migration inversely correlated with tumor necrosis-factor (TNF)-α and of EH-RV-LOEC tube formation with interferon (IFN)-γ (Figure 2S–T). Indeed, both the SDF-1 (CXCR4) and MCP-1 (CCR2) receptors were expressed on our patients’ LOEC (Figure S1 C–D).

Stratification based on statin treatment did not reveal significant differences between LOEC obtained from treated and untreated patients (Figure S3).

No other statistically significant correlations were detected.

Discussion

In the present study, we demonstrate that LOEC angiogenic activity is preserved in treated renovascular and essential hypertensive patients compared to normotensive controls. Moreover, LOEC expanded from either the renal vein or the systemic circulation showed comparable migratory, proliferative and tube-forming capacities to those HV-systemic-LOEC. These findings therefore support the use of ARAS and EH-LOEC for reparative purposes during renal injury.

We have previously found that the numbers of EPC were diminished in the systemic circulation of patients with EH or renovascular hypertension, and further reduced in the STK compared to the EH RV³. However, after 12 weeks of experimental renovascular hypertension in swine, LOECs showed unaltered proliferation and tube formation compared to normotensive pigs¹². Similarly, the present study shows that in patients with prolonged but medically-treated hypertension under controlled conditions, LOECs expanded from RV and systemic blood showed angiogenic capacity comparable to HV.

Previous studies have shown that hypertension attenuates the numbers of circulating EPC and colony forming units via increased tissue Ang-II¹³. Ang-II induces reactive oxygen species (ROS) production, which reduces EPC levels, impairs their function^{10, 14} and accelerates EPC senescence¹³. Inhibitors of the RAAS, like ACE-I or ARB, may have beneficial effects on the number and function of EPC in cardiovascular disease, including hypertension and atherosclerosis^{15, 16}. For example, Losartan decreased the expression of ROS-forming enzymes in the rat aorta, heart and kidney, resulting in increased EPC number and function¹⁵. Similarly, ARB improved the impaired EPC angiogenic capacity in EH patients¹⁷. In the present study, the angiogenic capacity of LOEC obtained from the RV or systemic blood in our hypertensive patients was not impaired compared with healthy controls, possibly because they were all treated with RAAS inhibitors. Additionally, the inverse correlation between LOEC function and blood pressure in EH patients suggested reduction of blood pressure as a potential mechanism by antihypertensive drugs enhance LOEC angiogenic capacity.

LOEC migration, proliferation and tube formation capacity in ARAS patients were also maintained. ARAS is characterized by renal hypoperfusion, activation of Ang-II, increased ROS generation and microvascular loss, as well as vascular wall and microvascular remodeling¹⁸. Coexistence of atherosclerosis and persistent hypoperfusion in ARAS kidneys magnifies oxidative stress and inflammation. As observed in other chronic kidney diseases, RAAS inhibitors attenuate oxidative stress and inflammation¹⁹, slow the progression of microvascular remodeling, and restore EPC levels and function^{15, 20}. Collectively, these effects may account for the preserved LOEC function in ARAS patients. Contrarily, this strategy was less effective in sustaining circulating EPC numbers that decline in such patients³, suggesting a different regulatory mechanism of EPC number and function.

Notably, most of our hypertensive patients were treated with statins and calcium-channel-blockers, which might also attenuate EPC apoptosis and increase EPCs mobilization and differentiation^{21, 22}. Thus, their pleiotropic effects may have also improved LOEC function. However, this similar relationship between LOEC and renal function in patients stratified by statin treatment argues against its effect on this relationship. Effects of other drugs will need to be tested in future studies designed for this purpose.

The type of EPC may also determine their functionality. Giannotti et al have shown that EOEC obtained from patients with untreated prehypertension and hypertension, and expanded for 7 days, have impaired function⁹. We studied function of LOEC obtained from patients treated with antihypertensive drugs under dietary and therapeutically-controlled conditions, and with only mildly elevated systolic pressures. Hence, collectively, these results suggest that antihypertensive treatment in hypertensive patients may improve angiogenic function of circulating progenitor cells.

Nevertheless, despite medical treatment, we observed a significant relationship between LOEC function and single-kidney hemodynamics assessed using MDCT. The migration capacity of only RV-LOEC correlated with STK-GFR, and tended to correlate with cortical perfusion. Furthermore, we found direct correlations between the migratory activity of RV-LOEC and CLK cortical perfusion, and between their proliferation and CLK-GFR. These observations suggest that in the ARAS STK and CLK, hemodynamics and function are determinants of the angiogenic potency of reparative cells that transit within their circulation, possibly by virtue of the intra-renal inflammatory and pro-oxidant microenvironment. Interestingly, we conversely observed an inverse correlation between IVC or RV-LOEC migration and both cortical perfusion and GFR in EH patients. Possibly in the EH kidney, hyperfiltration produces a noxious microenvironment that impairs EPC function akin to that caused by ischemia in the STK. Yet these effects on EPC function are likely subtle, given that overall EPC function in ARAS and EH patients were not different than those in HV.

In response to renal injury in ARAS, the STK releases inflammatory cytokines and specific injury homing signals to recruit and retain circulating EPC to stimulate its reparative process^{3, 7}. We previously observed an inverse correlation between the number of EPC and RV level of TNF-α and IFN-γ in EH and ARAS patients^{3, 23}. The current study extends our previous observations and demonstrates a significant inverse relationship between LOEC function and TNF-α or IFN-γ in ARAS and EH patients. Conversely, the anti-inflammatory cytokine IL-10 showed a direct correlation with LOEC migration. Therefore, inflammatory cytokines may play a role in regulating LOEC angiogenic function. Furthermore, levels of GM-CSF, G-CSF and SDF-1 correlated with LOEC function in all groups. These homing factors are all involved in the recruitment and mobilization of circulating progenitor cells from the bone marrow^{24, 25}, partly by bolstering their function. These molecules and their cognate receptor expressed on progenitor cells mediate the homing and engraftment process. Indeed, the SDF-1 receptor CXCR4 was expressed on our patients’ EPC. Thus, homing signals and inflammatory cytokines may regulate LOEC angiogenic function. Notably, RV level of MCP-1 correlated directly with STK and CLK RV-LOEC function. Indeed, MCP-1 has been shown to induce stem-cell migration and recruitment²⁶, and the expression of its CCR2 receptor on EPC in this study poses this chemokine as a homing and retention factor in human ARAS.

Our study is limited by a relatively small sample size and cross-sectional nature. We also excluded patients with diabetes or serum creatinine levels >1.7 mg/dl, because of application of iodinated contrast for MDCT scanning. Therefore, changes of EPCs function in these patients should be determined in future studies. The cause and effect relationships between renal hemodynamics, GFR, inflammatory markers and progenitor cell function, and the effects of anti-hypertensive regimen on the relationship between LOEC and renal function also merit further investigation. In addition, the mechanism by which inflammatory mediators modulate LOEC function, and changes in EOEC function in our patient groups, all await further studies.

Perspectives.

Our results demonstrate preserved function of systemic and RV LOEC in medically-treated renovascular and essential hypertensive patients. The correlation between LOEC function and renal function may imply that the ambient microenvironment exerts small but potentially meaningful effects on the function of EPC that transit across the renal circulation. Moreover, the correlation between LOEC function and inflammatory markers and homing signals in both ARAS and EH patients suggest that inflammation may regulate EPC function. Identification of these effects may allow refining novel therapeutic options for patients with ARAS. Therefore, in medically-treated patients with mildly elevated SBP under well-controlled conditions, LOEC function is relatively maintained and may permit their utilization for autologous delivery for potential reparative applications in case of imminent renal injury.

Novelty and Significance.

What Is New?

Our study demonstrates preserved EPC angiogenic function in patients with atherosclerotic renal artery stenosis compared with essential hypertensive patients and healthy volunteers. Yet, EPC function correlated with renal vein levels of anti-inflammatory mediator and endothelial progenitor cell homing factors.

What Is Relevant?

The adequate angiogenic function underscores EPC as a viable therapeutic option in essential and renovascular hypertensive patients.

Summary

The relatively preserved EPC function supports the feasibility of using autologous EPCs as a therapeutic tool in essential and renovascular hypertensive patients. Furthermore, homing signals and inflammatory mediators may modulate EPC angiogenic function.