Continuous Flow Left Ventricular Pump Support and Its Effect

WHAT IS NEW?

One potential risk associated with long-term exposure to nonphysiological blood flow patterns associated with durable continuous-flow left ventricular assist device (CF-LVAD) unloading is coronary artery endothelium-dependent dysfunction. Our study is the first to evaluate coronary vascular function before and after CF-LVAD intervention in the same patients.
We observed no changes in coronary artery endothelium-dependent or vascular smooth muscle vasorelaxation in 11 patients after >200 days of CF-LVAD circulatory support.
In a subgroup of 6 patients with end-stage ischemic cardiomyopathy, endothelium-dependent vaso relaxation improved after CF-LVAD intervention.

WHAT ARE THE CLINICAL IMPLICATIONS?

Our results indicate that coronary artery endothelial and vascular smooth muscle function is not adversely affected by CF-LVAD intervention.
These findings are important for all CF-LVAD recipients but most importantly for patients with significant improvement of cardiac structure and function who are deemed eligible for device weaning. In the case these patients undergo successful left ventricular assist device weaning, they will require a well-functioning coronary circulation to maintain adequate cardiac function in response to fluctuating myocardial oxygen demands during daily activities.
Further clinical research should evaluate endothelial cell morphology, vascular redox changes, sphingolipid metabolism, arterial fibrosis, and additional indices of vasoreactivity in a larger data set of responders and nonresponders to CF-LVAD intervention.

Introduction

Continuous-flow left ventricular assist devices (CF-LVADs) initially were developed to bridge end-stage heart failure (HF) patients refractory to traditional therapeutic modalities to eventual heart transplantation or to serve as a lifetime destination therapy for individuals deemed ineligible for transplant. Clinical experience with CF-LVAD support has shown that in a significant subset of patients with nonischemic cardiomyopathy (NICM), and a smaller subset of patients with ischemic cardiomyopathy (ICM), CF-LVAD intervention can reverse the complex process of chronic left ventricular (LV) remodeling.^1–4 Remarkably, some responders attain cardiac recovery to an extent and for a duration that allows for eventual CF-LVAD removal, precluding the need for heart transplantation and eliminating the inherent risk of major complications that are associated with long-term mechanical circulatory support.^5–16 One potential adverse event of long-term CF-LVAD support is arterial endothelial cell dysfunction that could result in impaired vascular reactivity. Strong rationale exists to investigate this issue in the coronary circulation of CF-LVAD recipients.

After the implementation of CF-LVAD circulatory support, the pulsatile nature of the arterial flow pattern decreases dramatically. Pulsatile shear stress and cyclic strain of an appropriate magnitude are requisite to maintain endothelial cell homeostasis.¹⁷ In this regard, Patibandla et al¹⁸ documented disrupted morphology, exaggerated proliferation, and increased mRNA and protein expression of antioxidant genes in human endothelial cells exposed to a hemodynamic pattern in vitro designed to mimic the environment experienced by CF-LVAD recipients versus healthy controls.¹⁹ In vivo, coronary artery perfusion occurs predominantly during diastole, when the reduced transmural pressure gradient facilitates coronary artery dilation and resultant blood flow. Unloading via CF-LVAD lowers LV end-diastolic pressures, but there is no mechanical systole and diastole, and the LVAD outflow graft has strong potential to disturb the normal laminar flow pattern into the coronary ostia. Recently, Ambardekar et al²⁰ reported marked remodeling concurrent with increased fibrosis in coronary arteries from patients with HF supported by CF-LVADs versus nonsupported HF patients and healthy controls. While the authors speculated that these structural changes might be responsible for attenuated coronary flow reserve observed previously in CF-LVAD recipients versus healthy controls,²¹ impaired endothelium-dependent or vascular smooth muscle function also could play a role. Here, we tested the hypothesis that durable CF-LVAD circulatory support impairs coronary artery endothelium-dependent vasorelaxation.

Methods

The authors will make the data, methods used in the analyses, and materials used to conduct this research available on reasonable request.

Study Population

Patients (n=38) with clinical characteristics consistent with chronic end-stage HF with reduced ejection fraction who required circulatory support with a CF-LVAD were enrolled in this study. Individuals requiring LVAD support because of acute HF, defined as <3 months of duration of HF symptoms and with no evidence of LV dilation, were excluded,² together with patients with hypertrophic or infiltrative cardiomyopathies. Adult LVAD patients were enrolled at institutions comprising the Utah Transplantation Affiliated Hospitals Cardiac Transplant Program: University of Utah Hospital, Intermountain Medical Center and Salt Lake VA Medical Center. Seven donors with structurally and functionally viable hearts that were not suitable for transplant secondary to noncardiac-related reasons served as normal controls. The study was approved by the respective institutional review board of the participating centers (IRB 00061825: Metabolic Profile of Patients Who Recover Their Myocardial Function During Mechanical Unloading), and informed consent was provided by all patients.

Coronary arteries were obtained (see below) from 38 patients at the time of CF-LVAD implant. Of these, coronary arteries were collected from 11 individuals again at CF-LVAD explant, that is, after LVAD intervention, at the time of heart transplant. An exploratory subgroup analysis was completed after the 11 CF-LVAD recipients were stratified based on the etiology of their HF. ICM (n=6/11) was defined as an left-ventricular ejection fraction <40% together with ≥1 of the following: (1) a history of myocardial infarction or revascularization; (2) a history of angina or chest pain and evidence of scarring in noninvasive imaging studies corresponding to previous myocardial infarction; (3) the presence of >75% stenosis of the left main or proximal left anterior descending artery; or (4) the presence of >75% stenosis of >2 epicardial vessels in a patient with unexplained cardiomyopathy. Patients with an left-ventricular ejection fraction <40% and nonobstructive coronary artery disease without evidence of prior myocardial infarction or revascularization were considered to have NICM (n=5/11).² A schematic of our subject population is shown in Figure 1.

**Figure 1.** **Study participants.** Of 45 subjects participating in this study, arteries were obtained from 38 patients with end-stage heart failure with reduced ejection fraction (HFrEF) and 7 nonfailing donor controls. Of the 38 patients with HFrEF, arterial function was assessed in 22 with end-stage heart failure of nonischemic cardiomyopathy (NICM) and 16 patients with end-stage heart failure of ischemic cardiomyopathy (ICM). In 11 of 38 patients, arterial function was assessed pre- and post–continuous-flow left ventricular assist device (LVAD) intervention in 5 NICM and 6 ICM subjects.

Clinical Data Collection

The Utah Cardiac Recovery LVAD database was queried for patients' characteristics including demographics, comorbidities, medications, laboratories, hemodynamics, and cardiac echocardiography information ≈1 week preceding CF-LVAD implantation, and within 6 to 8 weeks after implantation, while on mechanical support. Obtaining invasive hemodynamic, echocardiographic, and laboratory measurements at these time points is standard operating procedure at our participating institutions.

Tissue Collection

Myocardial tissue was obtained from the LV apical core at CF-LVAD implant, that is, before CF-LVAD intervention, from 38 patients. Of these, tissue was acquired again after CF-LVAD intervention in 11 patients from an apical area at least 1.0 to 1.5 cm distant from the CF-LVAD inflow cannula to minimize inclusion of reactive tissue changes. Thus, arteries were isolated from tissue samples obtained before and after CF-LVAD intervention from 11 subjects after CF-LVAD support, at the time of heart transplantation. Transmural apical core biopsies also were acquired from 7 normal hearts that were not allocated for transplantation because of noncardiac reasons. Each transmural biopsy used for vascular function analyses was placed immediately in iced normal physiological saline solution (pH 7.35–7.40) in the operating room, while myocardial tissue for histological analyses was placed immediately in 10% buffered formalin.

Vascular Function Analyses

With the aid of a dissecting microscope, coronary arteries were isolated from transmural biopsy tissue blocks that were immersed in iced normal physiological saline solution and pinned to a sylgard lined dissecting dish affixed to an ice pack. Epicardial coronary arteries were traced transmurally until second- and third-order branches were located and dissected free from adherent tissue. Vascular function was assessed in 1 to 4 coronary artery segments per time point per subject using isometric tension procedures. Resting arterial diameters and vessel lengths are reported in the respective figure legends. First, a series of internal circumference-active tension curves was completed on each artery to determine the vessel diameter that evoked the greatest tension development (ie, L_max) to 100 mmol/L potassium chloride. Second, arteries were precontracted to ≈65% of maximal potassium chloride–evoked tension development using the thromboxane A₂ receptor mimetic U46619. On stable tension development, responses to the endothelium-dependent vasodilator BK (bradykinin; 10⁻⁶–10⁻ ¹⁰ M) were recorded. Third, after arteries were precontracted to ≈65% of maximal potassium chloride–evoked tension development, responses to the endothelium-independent vasodilator sodium nitroprusside (SNP; 10⁻⁹–10⁻⁴ M) were assessed. At least 30 minutes separated each protocol. Results from multiple coronary artery segments per time point per subject were averaged to yield n=1. No evidence of atherosclerotic plaque was observed in any of the arteries that were evaluated. All data were recorded continuously using an analog-to-digital interface card (Biopac Systems, Inc, Santa Barbara, CA) that allowed for subsequent off-line quantitative analyses. We have described these procedures in arteries from humans,^22–25 rodents,^26–31 and pigs.³²

Histological Analyses: Whole-Field Digital Microscopy

After collection in the operating room, myocardial tissue for histological analyses remained in 10% buffered formalin for 24 hours. Next, tissue samples were dehydrated in increasing concentrations of alcohol, cleared through xylene, and subsequently embedded in paraffin. Tissue sections were cut in 4-μm sections and mounted on glass slides. To evaluate collagen content, tissue was stained with Masson Trichrome and was quantified by state-of-the-art whole-field digital microscopy (Aperio Technologies, Vista, CA).^33,34 The staining color threshold of the ImageScope 10.0 colocalization analysis algorithm (Aperio Technologies) was set to accurately identify collagen based on its blue color. Interstitial fibrosis was defined as the collagen content determined in the interstitial spaces and endomysial/perimysial spaces including the collagen content around capillaries and small vessels (ie, <60 µm internal diameter) found within those spaces. Total collagen content (ie, total fibrosis) was determined by including in our analysis the whole-field stained tissue, excluding only perivascular fat and a ≈1-mm border around the edges to exclude staining artifact. To evaluate microvascular density, the ImageScope 10.0 microvessels analysis algorithm was used to distinguish endothelial cells via transmembrane phosphoglycoprotein encoded by the CD34 gene immunostaining from nonspecific staining of surrounding tissue by applying appropriate dark and light thresholds. Only myocardial tissue cuts oriented in cross section (epicardium to endocardium) were analyzed. Microvascular density was defined as number of microvessels/total tissue analysis area. We have documented the above described methodologies.^33,34

Echocardiography

Transthoracic echocardiographic examination was performed at the laboratories of the participating institutions and was stored digitally. The studies included complete 2-dimensional, M-mode, and Doppler examinations. LV wall thickness, internal dimensions, and LV ejection fraction were obtained from standard views in accordance with current American Society of Echocardiography guidelines.³⁵

Statistical Analyses

Values are expressed as mean±SEM. Comparison of 1 end point (eg, microvascular density) pre- versus post–CF-LVAD intervention was performed using a paired t test. The comparison of multiple data points (eg, 10⁻ ⁶–10⁻¹⁰ M BK) obtained pre- versus post–CF-LVAD intervention was made using a repeated measures 2-way ANOVA. Tukey post hoc tests were performed when significant main effects were identified. Significance was accepted when P<0.05.

Results

Characteristics of the Study Populations

Characteristics of the 11 subjects wherein vascular and histological analyses were evaluated pre- and post–CF-LVAD intervention are shown in Tables 1 and 2, whereas characteristics of the 7 donor controls are shown in Table 3. Of the variables measured, CF-LVAD recipients were older, had lower ejection fraction, and greater LV end-diastolic dimension (all P<0.05) versus results obtained from the nonfailing donor controls. Comparing pre- to post–CF-LVAD intervention in all patients, systolic and diastolic pulmonary artery pressure, pulmonary capillary wedge pressure, pulmonary vascular resistance, and LV end-diastolic dimension decreased (P<0.05), cardiac output and cardiac index increased (P<0.05), and ejection fraction was not different. Further, while HbA1c (hemoglobin A1c) decreased pre- to post–CF-LVAD intervention (P<0.05), Na, Hb, blood urea nitrogen, and glucose were unaltered.

**Table 1.** Characteristics of All Patients Pre- and Post–Continuous-Flow LVAD Intervention
	LVAD Intervention: All (n=11)			LVAD Intervention: ICM (n=6)			LVAD Intervention: NICM (n=5)
	Pre	Post	P Value	Pre	Post	P Value	Pre	Post	P Value
Patient characteristics
Age, y	54±4	…		56±2	…		51±7	…	0.798
M/F	11/0	…		6/0	…		5/0	…
BMI, kg/m²	28.2±1.3	…		28.9±1.2	…		27.3±2.6	…	0.835
History of smoking	6/11	…		4/6	…		2/5	…
History of hypertension	4/11	…		4/6	…		0/5	…
D/ND	3/8	…		4/2	…		0/5	…
Hemodynamics
SBP, mm Hg	104±4	98±3	0.184	110±6	100±3	0.202	96±2	94±6	0.722
DBP, mm Hg	68±3	73±3	0.297	67±4	75±4	0.229	70±5	70±6	0.948
PA(S), mm Hg	55±3	40±4	0.001	54±5	44±6	0.022	56±5	34±4	0.006
PA(D), mm Hg	27±2	19±2	0.029	25±2	21±3	0.028	30±4	15±2	0.029
PCWP, mm Hg	25±8	14±7	0.011	21±6	17±7	0.376	30±8	10±5	0.028
PVR, Woods	4.5±0.7	3.0±0.4	0.009	4.9±1.0	3.6±0.6	0.022	4.1±1.1	2.2±0.3	0.017
CO, L/min	3.1±0.2	4.7±0.3	0.002	3.2±0.2	4.3±0.4	0.045	2.9±0.5	5.4±0.3	0.032
CI, L/min/m²	1.45±0.11	2.35±0.15	0.001	1.51±0.11	2.17±0.20	0.010	1.40±0.21	2.58±0.21	0.043
EF, %	16.0±1.9	16.6±1.2	0.322	17.7±3.3	19.3±0.7	0.526	14.0±1.4	15.0±1.5	0.276
LVEDD, cm	6.77±0.24	6.57±0.34	0.032	6.63±0.09	5.90±1.41	0.050	6.88±0.44	6.84±0.41	0.393
Serum
Na, mmol/L	135.0±2	138.2±0.5	0.099	137.7±1.7	138.2±0.6	0.749	131.8±2.9	138.2±0.9	0.098
Hb, g/dL	12.4±1	12.0±0.5	0.554	13.5±0.9	12.9±0.5	0.631	11.1±1.0	10.9±0.7	0.779
BUN, mg/dL	31.2±4	25.4±4.1	0.264	35.2±1.7	34.0±5.3	0.848	26.4±7.7	15.0±1.3	0.240
Glucose, mg/dL	123.8±10	116.8±10.3	0.577	120.0±14.0	126.0±8.0	0.757	128.4±17.4	105.8±8.8	0.161
HbA1c, %	7.1±0.5	5.7±0.3	0.012	7.7±0.6	5.9±0.3	0.034	6.1±0.4	No data

**Table 2.** LVAD Type and Patient Medication Usage
	LVAD Intervention: All (n=11)		LVAD Intervention: ICM (n=6)		LVAD Intervention: NICM (n=5)
	Pre	Post	Pre	Post	Pre	Post
LVAD type
HeartMate2	7/11		4/6		3/5
Jarvik2000	2/11		1/6		1/5
HeartWare	2/11		1/6		1/5
Medications
Inotropic	7/11	0/11	4/6	0/6	3/5	0/5
β-Blockers	8/11	6/11	5/6	3/6	3/5	3/5
ACE inhibitors	7/11	3/11	4/6	2/6	3/5	1/5
Aldosterone blockers	6/11	3/11	3/6	1/6	3/5	2/5
Diuretic	10/11	11/11	6/6	6/6	4/5	5/5
Acetylsalicylic acid	8/11	11/11	5/6	6/6	3/5	5/5
Clopidogrel	3/11	0/11	3/6	0/6	0/5	0/5
Anticoagulants	6/11	11/11	4/6	6/6	2/5	5/5
Antiarrhythmics	6/11	4/11	3/6	2/6	3/5	2/5
Statins	8/11	8/11	6/6	6/6	2/5	2/5

**Table 3.** Characteristics of Donor Controls
Age, y	45±3
M/F	1/6
D/ND	0/7
SBP, mm Hg	127±3
DBP, mm Hg	83±4
EF, %	60±2
LVEDD, cm	4.0±0.2
Glucose, mg/dL	122±10
HbA1c, %	5.4±0.1

Of the 11 patients wherein vascular and histological analyses were completed before and after CF-LVAD intervention, 5 fulfilled criteria to be classified as NICM, whereas 6 were categorized as ICM.² After stratification, NICM and ICM patients displayed demographic, hemodynamic, echocardiographic, and circulating characteristics similar to those described for the entire group.

Coronary Endothelial Function Is Impaired in Patients With End-Stage HF Caused by ICM

Endothelium-dependent vasorelaxation to BK was attenuated (P<0.05) in 28 coronary arteries obtained from 16 patients with ICM, versus 48 arteries obtained from 22 patients with NICM and 27 arteries obtained from 7 donor controls (Figure 2A). Endothelium-independent responses to SNP were similar among groups (Figure 2B).

**Figure 2.** **Coronary endothelial function is impaired in ischemic cardiomyopathy (ICM) patients vs nonischemic cardiomyopathy (NICM) patients and donor controls.** Endothelium-dependent vasorelaxation to BK (bradykinin) was attenuated in 28 coronary arteries (205±18 µm internal diameter (i.d.); 1700±48 µm length) obtained from 16 patients with ICM vs 48 arteries (204±14 µm i.d.; 1780±45 µm length) obtained from 22 patients with NICM and 27 arteries (181±13 µm i.d.; 1898±47 µm length) obtained from 7 nonfailing donor controls. B, Endothelium-independent responses to sodium nitroprusside were similar among groups. Total fibrosis (C; P=0.3544), interstitial fibrosis (D; P=0.6727), and microvascular density (E; P=0.4674) were similar in 22 patients with NICM vs 16 patients with ICM. Values are mean±SEM. Representative images of these data are shown in Figure I in the Data Supplement. HF indicates heart failure. *P<0.05 vs ICM.

Coronary Endothelial Function Is Not Altered by Durable CF-LVAD Circulatory Support

Our primary hypothesis was that coronary endothelial function would be impaired after implementation of durable CF-LVAD circulatory support. Of the 38 patients with end-stage HF wherein vascular function was evaluated, additional analyses were possible in 11 individuals 219±37 days after CF-LVAD intervention. BK- (Figure 3A) and SNP (Figure 3B)-evoked concentration-response curves were similar in 23 arteries obtained before versus 29 arteries obtained after the CF-LVAD intervention. These findings did not support our hypothesis.

**Figure 3.** **Coronary endothelial function is similar pre- and post–left ventricular assist device (LVAD) intervention.** A, BK (bradykinin)- and (B) sodium nitroprusside–evoked responses were similar in 23 arteries obtained before (197±22 µm internal diameter [i.d.]; 1572±83 µm length) vs 29 arteries obtained after continuous-flow left ventricular assist device (CF-LVAD) intervention (296±29 µm i.d.; 1880±29 µm length) in 11 patients. Total fibrosis (C; P=0.7827), interstitial fibrosis (D; P=0.7648), and microvascular density (E; P=0.0906) were similar before and after CF-LVAD intervention in these 11 patients. Values are mean±SEM. Representative images of these data are shown in Figure II in the Data Supplement.

Coronary Endothelial Function Is Improved by CF-LVAD Circulatory Support in Patients With ICM

Of the 11 patients wherein the impact of CF-LVAD intervention on coronary function was evaluated, an exploratory subgroup analysis was performed according to the etiology of their HF, that is, either NICM or ICM. In 6 ICM patients, BK (Figure 4A)-evoked vasorelaxation 256±54 days after CF-LVAD intervention was greater (P<0.05) in 14 coronary artery segments versus responses from 12 vessels obtained before the intervention. Responses to SNP were similar before and after LVAD intervention in arteries from ICM patients (Figure 4B). These results suggest that impaired BK-induced vasorelaxation observed before CF-LVAD intervention likely is secondary to compromised endothelial function in patients with ICM.

**Figure 4.** **Coronary endothelial function is improved by continuous-flow left ventricular assist device (CF-LVAD) circulatory support in ischemic cardiomyopathy (ICM) patients.** In 6 ICM patients, BK (bradykinin)-evoked vasorelaxation (A) after CF-LVAD intervention was greater in 14 coronary artery segments (260±34 µm internal diameter [i.d.]; 1833±50 µm length) vs responses obtained before the CF-LVAD intervention from 12 vessels (237±37 µm i.d.; 1833±50 µm length). Responses to sodium nitroprusside (B) were similar in arteries from ICM patients before and after CF-LVAD intervention. Total fibrosis (C; P=0.3319), interstitial fibrosis (D; P=0.1391), and microvascular density (E; P=0.1320) were similar before and after CF-LVAD intervention. Values are mean±SEM. Representative images of these data are shown in Figure III in the Data Supplement. *P<0.05 before vs after CF-LVAD intervention.

Coronary Endothelial Function Is Similar Before and After CF-LVAD Intervention in Patients With NICM

In 5 NICM patients, BK- (Figure 5A) and SNP (Figure 5B)-evoked concentration-response curves were similar in 11 arteries obtained before versus 15 arteries obtained 154±57 days after CF-LVAD intervention. These results indicate that endothelial and vascular smooth muscle function are not impaired by CF-LVAD placement in patients with HF of NICM origin.

Histological Analyses

Microvascular density, total fibrosis, and interstitial fibrosis was not different in patients with end-stage HF of ICM (n=16) or NICM (n=22) origin (Figure 2C through 2E; Figure I in the Data Supplement). Likewise, these end points were similar before and after CF-LVAD intervention in all patients (n=11; Figure 3C through 3E; Figure II in the Data Supplement), NICM patients (n=5; Figure 4C through 4E; Figure III in the Data Supplement), and ICM patients (n=6; Figure 5C through 5E; Figure IV in the Data Supplement).

Discussion

Here, we tested the hypothesis that CF-LVAD intervention impairs coronary artery endothelium-dependent vasorelaxation. Three main findings were observed. First, coronary endothelial function was impaired in patients with end-stage HF caused by ICM, compared with results obtained from patients with NICM and nonfailing donor controls (Figure 2A). Second, and contrary to our primary hypothesis, BK- and SNP-mediated vasorelaxation were similar in coronary arteries evaluated before and ≈220 days after CF-LVAD circulatory support (Figure 3A). Third, coronary artery endothelium-dependent dysfunction observed in ICM patients was less severe after CF-LVAD circulatory support (Figure 4A). Our results are important for CF-LVAD recipients in general and to the small percentage of responders (ie, patients with significant improvement of cardiac function) deemed eligible for LVAD device explant in particular. In this regard, explanted patients will require a viable coronary circulation to maintain myocyte function in response to fluctuating myocardial oxygen demands encountered during activities of daily living.

Peripheral Circulation Displays Heterogeneous Responses to CF-LVAD Unloading

Our primary hypothesis was based on rationale provided from previous investigations of the peripheral circulation,^36–38 coronary circulation,^16,21 and endothelial cell.^18,19 Regarding the peripheral circulation, Amir et al³⁶ documented reduced brachial artery flow-mediated vasodilation (FMD) in end-stage HF patients supported by CF versus pulsatile LVAD for ≈120 days. The authors speculated that lower pulsatile shear stress likely contributed to endothelial dysfunction, and Witman et al provide solid evidence to support this notion. A significant and positive relationship was observed between the pulsatility index and brachial artery flow-mediated vasodilation across healthy controls, class III/IV HF with reduced ejection fraction patients, and class III/IV patients supported by CF-LVAD.³⁷ Specifically, impaired FMD observed in class III/IV patients (pulsatility index, ≈8 arbitrary units) versus controls (pulsatility index, >12 arbitrary units) was further compromised in CF-LVAD recipients (pulsatility index, ≈2) after ≈150 days.³⁷ In contrast to findings from Witman et al, Sansone et al³⁸ reported that brachial artery FMD responses were attenuated similarly in end-stage HF patients and CF-LVAD recipients, versus results obtained from healthy controls. Of note, data from Cornwell et al suggest that vascular responses to CF-LVAD support might be regionally heterogeneous. For example, cerebral blood flow across a range of blood pressures triggered by a clinically relevant sit and stand maneuver was similar between CF-LVAD recipients and age/sex-matched healthy controls.³⁹

When considering these results from the periphery, it is important to note that (1) vascular responses were compared between/among different groups of subjects; (2) vascular smooth muscle function was tested directly only by Amir et al; (3) FMD and cerebral autoregulation include vasodilatory mechanisms that are endothelium-dependent and independent; and (4) the decreased pulsatility in CF-LVAD patients results in baroreceptor unloading, increased sympathetic nerve activity, and a hyperadrenergic milieu that has strong potential to precipitate endothelial dysfunction. Taken together, from what is known currently in the peripheral circulation, it is challenging to precisely discern the influence of CF-LVAD unloading on the endothelium per se.

Impaired Coronary Flow Reserve and Increased Coronary Artery Fibrosis in Response to CF-LVAD Circulatory Support

Few studies have examined the coronary circulation of LVAD recipients.^8,21 Using oxygen-15-labeled water H₂ ¹⁵O positron emission tomography, Tansley et al²¹ documented that patients with end-stage HF after ≈317 days of LVAD unloading had severely blunted coronary flow reserve versus age- and sex-matched subjects. Specifically, after the LVAD was turned off for 15 minutes, adenosine increased coronary flow from 1.46 to 1.49 mL/g per min in LVAD recipients (P=nonsignificant) versus 1.09 to 3.56 mL/g per min in healthy controls (P<0.05). While elevated fibrosis, reduced arteriolar density, mechanical hindrance (eg, elevated left ventricular end-diastolic pressures), and endothelial dysfunction were listed by the authors as factors potentially contributing to the attenuated coronary flow reserve in CF-LVAD recipients, none of these end points was measured. However, evidence supporting a role for fibrosis as a potential contributing factor to impaired coronary flow reserve was recently provided by Ambardekar et al.²⁰ For example, marked adventitial collagen deposition was observed in epicardial arteries obtained from HF patients after ≈213 days of CF-LVAD support versus age-matched HF patients and donor controls. Regarding mechanical hindrance, Tansley et al documented that increases (P<0.05) in basal myocardial blood flow (mL/g per min) from 0.95 (LVAD on) to 1.46 (LVAD off) likely resulted from a reduced transmyocardial pressure gradient throughout the cardiac cycle, coupled with elevated myocardial oxygen demand, with the LVAD off. Thus, while coronary flow reserve is a clinically relevant measure, it is difficult to discern the precise contribution from the endothelium per se using this end point in the context of mechanical circulatory support devices.

Hemodynamic Pattern Associated With CF-LVAD Disrupts Indices of Endothelial Cell Function Evaluated In Vitro

A reductionist approach was used to assess the unique hemodynamic pattern experienced by CF-LVAD patients directly on human endothelial cells in vitro. Patibandla et al¹⁸ observed disrupted morphology, proliferation, and increased mRNA and protein expression of antioxidant genes in endothelial cells exposed to cyclical pressure, shear stress, and axial and radial stretch characteristics for 72 hours that mimic arterial hemodynamics of CF-LVAD recipients versus disease-free individuals.¹⁹ The authors speculated that the strong antioxidant response might be secondary to an oxidative stress stimulus and eventually result in endothelial dysfunction. However, our results do not support this speculation. Here, we observed no changes in coronary artery endothelium-dependent vasorelaxation in 11 patients after >200 days of CF-LVAD circulatory support and improved coronary endothelium-dependent vasorelaxation after CF-LVAD in 6 ICM patients.

Reduced NO Bioavailability in the Context of Mechanical Unloading: Impaired NO Generation or Exaggerated NO Destruction?

A common speculation for why brachial artery FMD is attenuated by CF-LVAD unloading centers on reduced shear-stress–mediated eNOS (endothelial NO synthase) activation and NO generation secondary to long-term exposure to continuous flow, but direct evidence for this mechanism is lacking.^36,37 Instead, however, Sansone et al³⁸ demonstrated that increased NO degradation might make an important contribution to arterial dysfunction observed in CF-LVAD patients. For example, elevated free hemoglobin and microparticles of red blood cell and endothelial cell origin were observed in the circulation of CF-LVAD recipients versus healthy controls, likely caused by hemolysis associated with high mechanical forces exerted by the CF-LVAD.^40–43 Because brachial artery FMD improved in CF-LVAD patients after receiving a heart transplant, in parallel with reduced circulating microparticles and return to a pulsatile flow pattern,³⁸ it is not unreasonable to speculate that mechanisms related to reduced NO generation and exaggerated NO degradation likely occur in a concurrent manner.

CF-LVAD Circulatory Support Does Not Impair Coronary Artery Endothelial Function

We tested our primary hypothesis by evaluating vascular function before and after CF-LVAD intervention in the same subjects. This sampling approach is ideal to assess the impact of long-term CF-LVAD support on arterial reactivity in a manner that has strong potential to limit intersubject variability. To evaluate endothelium-dependent function, we measured concentration-response curves to BK. BK is produced endogenously by the vascular kinin-kallikrein system and stimulates B₂ receptors to evoke vasodilation in the human coronary circulation.⁴⁴ Companion concentration-response curves were completed in the same arteries for every experiment using the endothelium-independent vasodilator SNP. Further, we examined responses to BK and SNP in coronary artery segments ex vivo using isometric tension procedures. This technique eliminates the contribution from neural, humoral, and mechanical influences that are known to impact vascular reactivity in vivo. This is an especially important consideration concerning the in vivo study of vascular function in CF-LVAD recipients because (1) baroreceptor unloading may precipitate a heightened vasoconstrictor milieu; (2) circulating factors associated with device-specific mechanical forces could exaggerate NO degradation; and (3) elevated left ventricular end-diastolic pressures can hinder coronary flow. Using the above approach and methodology, we noted that neither endothelial nor vascular smooth muscle function exhibited by coronary arteries was altered by CF-LVAD circulatory support, and these findings did not support our initial hypothesis (Figure 3A). Of surprise to us was the observation that coronary endothelial function displayed by patients with end-stage HF caused by ICM was greater after versus before the CF-LVAD intervention (Figure 4A). Improved BK-induced vasodilation does not appear to be associated with a responder phenotype (ie, LVAD patients exhibiting significant cardiac functional/structural improvement), as neither ejection fraction nor left ventricular end-diastolic dimension were different when assessed at both time points (Table 2).

Limitations

Several limitations to our study will be addressed. First, vascular function was assessed in a modest number of patients before and after CF-LVAD intervention. Because the error of measurement did not overlap when significant differences were obtained, we are confident in our results. Second, fibrosis was not assessed directly in the second- and third-order arterial branches wherein function was measured, but total fibrosis, interstitial fibrosis, and microvascular density were not different in myocardial tissue obtained before and after CF-LVAD intervention (Figures 3C through 3E, 4C through 4E, and 5C through 5E). Third, although the mean duration of CF-LVAD support was ≈220 days in our study, invasive hemodynamic-echocardiographic-laboratory measurements were assessed on all patients 6 to 8 weeks post-LVAD implantation (Table 1). Therefore, it is possible that changes could have occurred at time points distal to this measurement. Fourth, a significant reduction in HbA1c existed post- versus pre-LVAD intervention in ICM patients, yet such data were unavailable post-LVAD intervention in NICM patients. It is a possibility that greater reductions in HbA1c occurred in ICM versus NICM patients, and this improvement in the systemic milieu could have contributed to better endothelium-dependent vasorelaxation in arteries from ICM patients post- versus pre-LVAD intervention (Figure 4). Finally, the precise mechanism for greater endothelium-dependent vasorelaxation in patients with end-stage HF caused by ICM versus NICM after CF-LVAD intervention was not explored, but ongoing studies by our laboratory team are evaluating endothelial cell morphology, vascular redox changes, sphingolipid metabolism, arterial fibrosis, and additional indices of vasoreactivity in a larger data set of responders and nonresponders to CF-LVAD intervention in an effort to enrich our novel findings.

Sources of Funding

Funding was provided for Dr Symons by AHA16GRNT31050004, NIH RO3AGO52848, and NHLBI RO1 grant HL141540; L. Deeter, N. Deeter, and T. Bonn by the University of Utah Undergraduate Research Opportunities Program; J.M. Cho by a University of Utah Graduate Research Fellowship; and Dr Drakos by American Heart Association Heart Failure Strategically Focused Research Network Grant 16SFRN29020000, NHLBI RO1 grant HL135121, NHLBI RO1 grant HL132067 and the Nora Eccles Treadwell Foundation.

Footnotes

Guest Editor for this article was John C. Burnett, Jr, MD.

The Data Supplement is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCHEARTFAILURE.119.006085.

Current address for Dr Diakos: Tufts Medical Center Division of Cardiology, Boston, MA.

J. David Symons, PhD, Division of Endocrinology, Metabolism, and Diabetes, University of Utah, 15 N 2030 E, Bldg 533, Rm 3420, Salt Lake City, UT 84112, Email j.david. [email protected] utah.edu

Stavros G. Drakos, MD, PhD, Nora Eccles Harrison Cardiovascular Research and Training Institute, 95 S 2000 E, Salt Lake City, UT 84112, Email stavros. [email protected] utah.edu

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