A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

Summary

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

Abstract

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

Introduction

Heart failure is the leading cause of death in developed countries and is expected to increase by 25% over the next decade. Pulmonary hypertension (PH) – a pathological increase of blood pressure in the pulmonary circulation – affects approximately 70% of patients with end-stage heart failure; the World Health Organization classifies PH as pulmonary hypertension due to left heart disease (PH-LHD)¹. PH-LHD is initiated by impaired systolic and/or diastolic left ventricular (LV) function that results in elevated filling pressure and passive congestion of blood into the pulmonary circulation². Albeit initially reversible, PH-LHD gradually becomes fixed due to active pulmonary vascular remodeling in all compartments of the pulmonary circulation, i.e., arteries, capillaries, and veins^3,4. Both reversible and fixed PH increase RV afterload, initially driving adaptative myocardial hypertrophy but ultimately causing RV dilatation, hypokinesis, fibrosis, and decompensation that progressively lead to RV failure^1,2,5,6. As such, PH accelerates disease progression in heart failure patients and increases mortality, particularly in patients undergoing surgical treatment by implantation of left ventricular assist devices (LVAD) and/or heart transplantation^7,8,9. Currently, no curative therapies exist that could reverse the process of pulmonary vascular remodeling, so fundamental mechanistic research in appropriate model systems is needed.

Importantly, clinical studies show that PH-LHD as a frequent complication in patients with aortic stenosis can improve rapidly in the early post-operative period following aortic valve replacement¹⁰. Analogously, high (>3 Wood Units) pre-operative pulmonary vascular resistance (PVR) that was, however, reversible on nitroprusside was sustainably normalized after heart transplantation in a 5-year follow-up study¹¹. Similarly, an adequate reduction of both reversible and fixed PVR and improvement of RV function in LHD patients could be realized within several months by unloading the left ventricle using implantable pulsatile and non-pulsatile ventricular assist devices^12,13,14. Currently, the cellular and molecular mechanisms that drive reverse remodeling in the pulmonary circulation and RV myocardium are unclear. Yet, their understanding may provide important insight into physiological pathways that may be therapeutically exploited to reverse lung vascular and RV remodeling in PH-LHD and other forms of PH.

A suitable preclinical model that adequately replicates the pathophysiological and molecular features of PH-LHD can be used for translational studies in pressure overload-induced congestive heart failure due to surgical aortic banding (AoB) in rats^4,15,16. In comparison to similar heart failure due to pressure overload in the murine model of transverse aortic constriction (TAC)¹⁷, banding of the ascending aorta above the aortic root in AoB rats does not produce hypertension in the left carotid artery as the banding site is proximal of the outflow of the left carotid artery from the aorta. As a result, AoB does not cause left-sided neuronal injury in the cortex as is characteristic for TAC¹⁸, and which may affect the study outcome. Compared to other rodent models of surgically induced PH-LHD, rat models in general, and AoB in particular, prove to be more robust, reproducible and replicate the remodeling of the pulmonary circulation characteristic for PH-LHD patients. At the same time, perioperative lethality is low¹⁹. Increased LV pressures and LV dysfunction in AoB rats induce PH-LHD development, resulting in elevated RV pressures and RV remodeling. As such, the AoB rat model has proven extremely useful in a series of previous studies by independent groups, including ourselves, to identify pathomechanisms of pulmonary vascular remodeling and test potential treatment strategies for PH-LHD^{4,15,20,21,22,23,24,25}.

In the present study, the AoB rat model was utilized to establish a surgical procedure of aortic debanding to study mechanisms of reverse remodeling in the pulmonary vasculature and the RV. Previously, myocardial reverse remodeling models such as aortic debanding in mice²⁶ and rats²⁷ have been developed to investigate the cellular and molecular mechanisms regulating the regression of left ventricular hypertrophy and test potential therapeutic options to promote myocardial recovery. Moreover, a limited number of earlier studies have explored the effects of aortic debanding on PH-LHD in rats and showed that aortic debanding might reverse medial hypertrophy in pulmonary arterioles, normalize the expression of pre-pro-endothelin 1 and improve pulmonary hemodynamics^27,28, providing evidence for the reversibility of PH in rats with heart failure. Here, the technical procedures of the debanding surgery are optimized and standardized, e.g., by applying a tracheotomy instead of endotracheal intubation or by using titanium clips of a defined inner diameter for aortic banding instead of polypropylene sutures with a blunt needle^26,27, thus providing for better control of the surgical procedures, increased reproducibility of the model and an improved survival rate.

From a scientific perspective, the significance of the PH-LHD debanding model does not solely lie in demonstrating the reversibility of the cardiovascular and pulmonary phenotype in heart failure, but more importantly, in the identification of molecular drivers that trigger and/or sustain reverse remodeling in pulmonary arteries as promising candidates for future therapeutic targeting.

Protocol

All procedures were performed following the "Guide for the Care and Use of Laboratory Animals" (Institute of Laboratory Animal Resources, 8th edition 2011) and approved by the local governmental animal care and use committee of the German State Office for Health and Social Affairs (Landesamt für Gesundheit und Soziales (LaGeSO), Berlin; protocol no. G0030/18). First, congestive heart failure was surgically induced in juvenile Sprague-Dawley rats ~100 g body weight (bw) (see Table of Materials</strong…

Representative Results

First, successful aortic debanding was confirmed by transthoracic echocardiography performed before and after the debanding procedure in AoB animals (Figure 6). To this end, the aortic arch was assessed in parasternal long axis (PLAX) B-mode view. The position of the clip on the ascending aorta in AoB animals and its absence after the Deb surgery was visualized (Figure 6A,B). Next, aortic blood flow was evaluated by pulsed-wave Doppler…

Discussion

Here, a detailed surgical technique for aortic debanding in a rat AoB model is reported that can be utilized to investigate the reversibility of PH-LHD and the cellular and molecular mechanisms that drive reverse remodeling in the pulmonary vasculature and the RV. Three weeks of aortic constriction in juvenile rats results in PH-LHD evident as increased LV pressures, LV hypertrophy, and concomitantly increased RV pressures and RV hypertrophy. Aortic debanding at week 3 post-AoB was able to unload the LV and fully reverse…

Materials

Amoxicillin	Ratiopharm	PC: 04150075615985	Antibiotic
Anti-BNP antibody	Abcam	ab239510	Western Blotting
Aquasonic 100 Ultrasound gel	Parker Laboratories	BT-025-0037L	Echocardiography consumables
Bepanthen	Bayer	6029009.00.00	Eye ointment eye ointment
Carprosol (Carprofen)	CP-Pharma	401808.00.00	Analgesic
Clip holder	Weck stainless USA	523140S	Surgical instruments
Fine scissors Tungsten carbide	Fine Science Tools	14568-12	Surgical scissors
Fine scissors Tungsten carbide	Fine Science Tools	14568-09	Surgical scissors
High-resolution imaging system	FUJIFILM VisualSonics, Amsterdam, Netherlands	VeVo 3100	Echocardiography machine. Images were acquired with pulse-wave Doppler mode, M-mode and B-mode
Isoflurane	CP-Pharma	400806.00.00	Anesthetic
Ketamine	CP-Pharma	401650.00.00	Anesthetic
Mathieu needle holder	Fine Science Tools	12010-14	Surgical instruments
Mechanical ventilator (Rodent ventilator)	UGO Basile S.R.L.	7025	Volume controlled respirator
Metal clip	Hemoclip	523735	Surgical consumables
Microscope	Leica	M651	Manual surgical microscope for microsurgical procedures
Millar Mikro-Tip pressure catheters	ADInstruments	SPR-671	Hemodynamics assessment
Moria Iris forceps	Fine Science Tools	11373-12	Surgical forceps
Noyes spring scissors	Fine Science Tools	15013-12	Surgical scissors
Povidone iodine/iodophor solution	B/Braun	16332M01	Disinfection
PowerLab	ADInstruments	4_35	Hemodynamics assessment
Prolene Suture, 4-0	Ethicon	EH7830	Surgical consumables
Rib spreader (Alm selfretaining retractor blunt, 70 mm, 2 3/4″)	Austos	AE-BV010R	Surgical instruments
Serrated Graefe forceps	Fine Science Tools	11052-10	Surgical forceps
Silk Suture, 4-0	Ethicon	K871	Surgical consumables
Skin disinfiction solution (colored)	B/Braun	19412M07	Disinfection
Spectra 360 Elektrode gel	Parker Laboratories	TB-250-0241H	Echocardiography consumables
Sponge points tissue	Sugi	REF 30601	Surgical consumables
Sprague-Dawley rat	Janvier Labs, Le Genest-Saint-Isle, France		Study animals
Tracheal cannula			Outer diameter 2 mm
Xylazin	CP-Pharma	401510.00.00	Anesthetic