Pulmonary artery hypertension (PAH) represents a variety of conditions with similar features in terms of structural changes and clinical presentation, as it results from a chronic obstruction of small arterial pulmonary vessels. The severe involvement of the right ventricle (RV) can, then, lead to heart failure and, ultimately, to death.
In recent years, much more insight has been given to the pathogenic role of HIV in the development of PAH that currently represents one of the most severe events during the HIV disease. Indeed, an association between HIV and PAH has been clearly established in the most recent pulmonary hypertension classification .
Before the advent of HAART, HIV-PAH was often undetected due to the short course of HIV infection and the abundance and severity of opportunistic infection. With the introduction of HAART, which resulted in a longer survival and in a reduction of mortality, related to opportunistic infections and cancer in HIV-infected individuals, much more attention has been given to chronic manifestations including metabolic involvement, cardiovascular disease and pulmonary hypertension.
In 1991, Speich et al. estimated a 0.5% prevalence of PAH in the HIV population, which was 2500-fold greater than that observed in the general population (1–2 per million). This figure has not changed in the recent years; however, a decrease in the incidence has been found in the Swiss HIV Cohort Study, assessing the incidence of newly diagnosed cases of pulmonary hypertension in HIV-infected individuals, namely 0.24 and 0.03% in 1993 and 2006, respectively .
HIV-PAH occurs in early and late stages of HIV infection, without a relationship with the stage of the disease, the degree of CD4+ level and the risk factors for HIV acquisition. Patients with HIV-PAH are younger than those without PAH; men are more frequently affected by HIV-PAH, likely reflecting the high male prevalence in the HIV population. Mortality is significantly higher in HIV-PAH patients as compared to non-PAH HIV-infected individuals, and death is often related to heart failure rather than to other HIV complications. HAART seems to play a favourable role on the survival of these patients; however, prognosis is strongly related to the degree of cardiac involvement. Approximately 90% of patients with HIV-PAH survive for 1 year, whereas 70% have a 3-year survival rate [4,5].
Pathogenesis of PAH in HIV is still unclear and whether HIV infects lung vascular endothelial cells is still under discussion; however, viral proteins may promote apoptosis, growth, and proliferation of endothelial and smooth muscle cells . Recently, new insights have been described on the association between HIV negative factor F-protein (Nef) polymorphisms in functional domains and the HIV-PAH phenotype . Moreover, PAH has been associated with viral proteins in infections with hepatitis B  and human herpes virus (HHV)-8  that frequently co-infect HIV-infected individuals.
Knowledge of myocardial structure and infections in patients with HIV-PAH can be of help to recognize the mechanisms that are involved in the right ventricular remodelling and functional deterioration as well as to analyse the coronary microcirculation as a possible model for the interpretation of micro-vascular pulmonary disease.
To this regard, a systematic cardiac biopsy study, to our knowledge, has never been obtained, probably because of rarity of the disease and the risk of HIV exposure for healthcare workers. In the present study, the results of a bi-ventricular endomyocardial biopsy study evaluating the histological changes and the myocardial infectious profile of patients with HIV-associated PAH and right ventricular dysfunction are presented.
Among 300 patients with PAH referred between January 2007 and December 2012 to our centre for diagnostic evaluation and treatment, 37 patients were affected by HIV infection, 25 of them had an associated compromise of right ventricular function and 15 (10 men, mean age 46 ± 6 years) accepted to have an invasive cardiac study with endomyocardial biopsy in the attempt to identify the cause and mechanism of cardiac deterioration. This last cohort constituted the population of our study. All cardiac studies were approved by the Ethics Committee of our Institution, and all patients gave their informed consent. The 22 patients with PAH and HIV infection that did not participate in the study in 12 cases had normal right ventricular function so that the invasive cardiac study was not proposed, and 10 patients did not agree to participate in the study. These last 10 patients did not differ from the patient population for age, sex, HIV status and degree of PAH.
All patients had ECG, Holter monitoring, two-dimensional (2D)-echocardiogram with Doppler analysis and cardiac magnetic resonance (CMR) with gadolinium infusion using a 1.5-T scanner (Siemens Avanto, Erlangen, Germany). CMR acquisition protocol included standard sequences for the evaluation of biventricular volumes and function (cine steady-state free precession), myocardial oedema (T2-weighted short tau inversion recovery) and late enhancement after intravenous injection of 0.1 mmol/kg of gadolinium (Gd-BOPTA; Multihance, Bracco, Milan, Italy; inversion-recovery contrast-enhanced sequences). Suspicion of myocardial inflammation was raised in presence of a combination of tissue oedema and late enhancement abnormalities with non-ischemic pattern of distribution (meso-epicardial linear striae) .
All patients underwent right and left cardiac catheterization, coronary with ventricular angiography and bi-ventricular endomyocardial biopsy.
Endomyocardial biopsies were performed in the septal–apical region of both ventricles, which were approached by a 7-F (501–613A Cordis) long sheet and identified on an X-ray view using flashing of contrast medium. Three to four endomyocardial samples of each ventricular chamber were drawn, cut and processed for histology, immunohistochemistry and molecular biology studies. Controls for histology, immunohistochemistry and molecular studies were surgical biopsies from papillary muscles of 10 patients with mitral stenosis and normal left ventricular dimension and function, undergoing valve replacement. These endomyocardial fragments, although not obtained from healthy individuals, were derived from a not overloaded chamber and were considered the nearest samples to a normal endomyocardial tissue .
Histological and immunohistochemical studies
Two/three endomyocardial biopsy specimens from each ventricle were fixed in 10% buffered formalin, embedded in paraffin wax, cut into 5-μ thick sections and stained with haematoxylin–eosin, Masson trichrome and Miller's elastic Van Gieson. The diagnosis of myocarditis was reached by a pathologist blind to clinical data, according to Dallas criteria , and confirmed by immunohistochemical characterization of inflammatory cells (anti-CD45, CD43, CD45RO, CD20, CD8, CD4, CD31, CD68 antibodies; Dako, Carpinteria, California, USA). In particular, the presence of more than 14 infiltrating leukocytes per mm2, often adherent to the contour of cardiomyocytes and focally associated with cell necrosis, were considered diagnostic for active myocarditis. Immunohistochemistry on paraffin sections was also obtained for assessment of cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) (P3C4; Santa Cruz Biotechnology, California, USA) by immunofluorescence and E-selectin (D-7; Santa Cruz Biotechnology), intercellular adhesion molecule-DR (ICAM-1) (Santa Cruz Biotechnology), human leukocyte antigen-DR (HLA-DR) (Dako) by immunoperoxidase, in order to study endothelial inflammatory activation in coronary small vessels. Semi-quantitative evaluation of immunostaining was graded as follows: grade 0, no discernible immunoreactivity; grade 1, weak staining; grade 2, moderate expression; and grade 3, strong immunoreactivity. VCAM-1 and E-selectin, known to be normally absent and induced during inflammation, were scored positive when immunostaining exceeded grade 0, whereas ICAM-1 and HLA-DR that are constitutively expressed were scored positive if immunoreactivity exceeded grade 1 . Immunohistochemical localization of viral antigen specific for the HCV c100 protein (TORDJI-22; Biogenex Laboratories, San Ramon, California, USA)  and for adenovirus (Novus Biologicals, UK) was performed in PCR-positive patients to investigate vascular and cardiomyocyte infectious involvement.
Molecular biology studies
PCR and reverse transcription (RT)-PCR analysis was performed on two frozen endomyocardial biopsy tissue samples to search for HIV and for the most common DNA [adenovirus, cytomegalovirus (CMV), parvovirus B19, Epstein–Barr virus (EBV), HHV-6 and HHV-8, herpes simplex virus 1 and 2] and RNA (enterovirus, influenza virus A and B, hepatitis C virus (HCV)] cardiotropic viruses as described .
Normal distribution of variables was assessed with Kolmogorov–Smirnov test. Quantitative measurements were expressed as mean + SD. Categorical data were presented as absolute frequencies and percentage values. Difference between two groups was determined by unpaired t test for continuous variables and Fisher's exact test for categorical data. A two-tailed P value less than 0.05 was considered statistically significant. Computations were performed with SPSS 20 (IBM, Armonk, New York, USA).
Clinical characteristics with histological and molecular findings of the 15 HIV-PAH patients studied are summarized in Table 1. Risk factors for HIV infection were drug use (12 patients), transfusion (2 patients) and unknown (1 patient). Average nadir CD4+ cell count was 394 cell/μl (range 150–1255). HIV-RNA was undetectable in 14 out of 15 patients, and was 2297 copies/ml in one patient. Time on HAART was on average 10.2 years (range 3–20.5). HIV immune status according to Centers for Disease Control and Prevention (CDC) classification was A3, B1, B2, B3, and C1 in two, three, two, three, and five patients, respectively (Table 1).
Cardiac magnetic resonance imaging
Cardiac magnetic resonance documented an increased right ventricular end-diastolic volume (mean 232 ± 75 ml) with reduced right ventricular ejection fraction (mean 32 ± 9%). Left ventricular end-diastolic volume (LVEDV) (mean 109 ± 18 ml) as well as left ventricular ejection fraction (LVEF) (mean 57 ± 5%) were in the normal range. Subepicardial and/or mesocardial delayed enhancement in association with myocardial oedema was observed in the inter-ventricular junction and/or left ventricular infero-lateral free wall denoting myocarditis (Fig. 1 a and b) in eight out of 15 patients (60%).
Haemodynamic and angiographic findings
All patients had a remarkable increase pulmonary artery pressure (PAP) (mean value 50 ± 10 mmHg) with a normal wedge pressure (mean value 9 ± 2 mmHg). Right ventricular end-diastolic pressure (EDP) was always elevated (>10 mmHg), whereas left ventricular EDP (LVEDP) was within normal limits (<12 mmHg). These haemodynamic changes were associated with an increased New York Heart Association (NYHA) class of 2.4 ± 0.5. Ventricular angiography showed a dilated and hypokinetic RV with various degrees of tricuspid incompetence; the left ventricle (LV) had normal volumes, but in eight cases, micro-aneurysms of inferior and/or apical left ventricular wall and/or inferior right ventricular wall were observed (Fig. 2 a–d). Coronary angiography in all patients showed a structurally normal network with a slow run-in and run-off of contrast medium.
Histology and immunohistochemistry
Inflammatory infiltrates focally associated with necrosis of the adjacent cardiomyocytes were documented in 12 out of 15 HIV-associated PAH patients (Fig. 1c). Myocardial inflammation was detectable in positive cases in both RV and LV biopsy samples. Interstitial and replacement fibrosis was usually present, suggesting a chronic inflammatory process. Inflammatory infiltration of some arterioles’ wall was observed in three patients with micro-aneurysms at ventricular angiography (Fig. 2e). In the remaining three patients, an increased interstitial and replacement fibrosis was observed, suggesting the possibility of a healed myocarditis.
Immunohistochemical quantitation of adhesion molecules documented an increased expression of VCAM-1 (1.9 ± 6.1 in patients vs. 0 in controls; P < 0.001), E-selectin (1.7 ± 07 in patients vs. 0 in controls), ICAM-1 (2.3 ± 0.5 in patients vs. 0.8 ± 0.4 in controls) and HLA-DR (1.8 ± 0.8 in patients vs. 0.8 ± 0.4 in controls; P < 0.001) in the endothelial layer of intramural vessels compared with controls (Fig. 3). In particular, VCAM-1 and E-selectin were absent in normal controls, whereas ICAM-1 and HLA-DR were only weakly expressed.
Immunohistochemical assessment of viral proteins showed a positivity of adenovirus antigen (Fig. 1d) and HCV core antigen TORDJI-22 (Fig. 2f) in cardiomyocytes and in vessel wall of PCR-positive patients.
Molecular biology studies
Myocardial PCR was positive in six patients (Table 1) – two for adenovirus infection (Fig. 1c, square) and four for HCV infection (Fig. 1e, square), whereas it was negative for HIV in all patients. In adenovirus patients, PCR in blood samples was negative for the same virus, whereas a remarkable lower HCV viral load was observed in HCV-infected patients.
Occurrence of PAH adversely affects the prognosis of patients with HIV infection, with decline of survival rate to 70% at 3 years and death being mainly related to heart failure rather than to other HIV complications [4–5]. In this context, preservation of right ventricular function is crucial as its deterioration is usually harbinger of grim outcome. In pathogenetic terms, right ventricular remodelling and dysfunction are usually attributed to severity of PAH and of pressure overload, so that treatment of such patients is essentially directed to lowering and controlling PAP by use of powerful vasodilators like sildenafil  and the endothelin receptor inhibitor bosentan . The present study shows that inflammation of myocardium and intramural vessels is detectable in most patients with right ventricular dysfunction and that it may contribute to cardiac deterioration and heart failure.
Possible presence of myocarditis was raised by CMR showing in HIV-PAH patients with right ventricular dysfunction, signal abnormalities as oedema and delayed enhancement after gadolinium infusion affecting subepicardial and/or mesocardial layers. Ventricular angiography added the visualization of left ventricular and/or right ventricular micro-aneurysms in 53% of the patient population that in the absence of coronary artery disease are commonly referable  to myocardial inflammation. The high incidence of cardiac micro-aneurysms testifies of severity of myocarditis as well as of associated involvement of myocardial vessels. Low CMR sensitivity for detection of inflammatory micro-aneurysms has been already reported and should be taken into consideration, particularly in young patients with unexplained ventricular arrhythmias . Finally, histology showed a myocarditic process in 12 out of 15 patients studied, with microvasculitis being documented in three cases. The diagnosis was reached by a pathologist blind to clinical data applying ‘Dallas criteria’, implemented by immunohistochemical characterization of inflammatory cells. Remarkably, an enhanced expression of adhesion molecules was found in the endothelium of all patients, suggesting a common inflammatory involvement of intramural vessels. This last aspect may reflect in an extensive endothelial dysfunction  and explain the slow flow observed during coronary angiography. It can be argued that myocarditis can be a secondary event induced by PAH (through cardiomyocyte damage with activation of an autoreactive pathway) and that it should, anyway, have a minor role compared with increased PAP, because of discrepancy between RV and LV compromise. Indeed LV in PAH patients is under-perfused and the normal values of left ventricular filling pressure as well as the major thickness of the left ventricular wall can mask the severity of left ventricular involvement. On the contrary, micro-aneurysms were observed in both ventricles and histological evidence of myocarditis was documented, in positive cases, in both right ventricular and left ventricular endomyocardial biopsy samples.
Finally, viral studies, obtained by PCR on two tissue samples and using a large panel of primers, documented the presence of infectious agents including HCV and adenovirus in 40% of cases and viruses localized in both cardiomyoytes and intramural vessels. Parvovirus B19, commonly reported in microvascular dysfunction by German authors , was not detected in our series, likely suggesting a different epidemiologic context.
Overall, the findings observed in this study suggest an increased susceptibility of HIV-infected patients to myocardial viral infection and to myocardial and microvascular inflammation contributing to impairment of cardiac function and occurrence of major cardiovascular events as heart failure and sudden death.
Potential relationship between myocardial inflammation/infection and pulmonary artery hypertension
Autopsy studies in patients with HIV-associated PAH  failed to document clear-cut inflammation/infection of pulmonary microcirculation that appear characterized by non-specific lesions. However, PAH has been associated with viral proteins in infections with hepatitis B , hepatitis C  and HHV-8 , which frequently co-infect HIV-infected individuals. In particular, in patients with HHV-8 infection and PAH, viral protein latency-associated nuclear antigen (LANA)-1 has been detected in the cells of plexiform lesions in 62% of the cases. Moreover, viral proteins are known to promote apoptosis, necrosis, growth, and proliferation of endothelial and smooth muscle cells .
It is possible that persistent release of inflammatory cytokines and/or viral proteins from an inflamed/infected myocardium might induce an inflammatory damage of pulmonary vessels progressing by intimal fibrosis and smooth muscle cell proliferation to a common final pathway represented by plexiform vascular lesions. In support to this hypothesis is the evidence that the introduction of HAART has improved the survival of patients with HIV-associated PAH .
Recognition of myocarditis and microvascular inflammation may influence the prevention and treatment of right ventricular dysfunction in patients with HIV-associated PAH. In particular, virus-positive myocarditis may benefit from beta-interferon administration in case of enterovirus and adenovirus infection ; ribavirin in combination with steroids can be adopted in HCV infection , and the advent of direct acting antivirals with high cure rates  could have a greater impact on incidence and outcomes; acyclovir may be used in Herpes virus infection and gancyclovir may affect CMV myocardial infection. Immune-modulating therapy, including high-dose immunoglobulins, immunoadsorption  and immunosuppression , may have a role in the control of virus-negative myocarditis. In addition, if inflammatory damage of pulmonary vessels co-exists, antiviral or immunomodulating therapy could result in halting or improvement of PAH severity.
In conclusion, inflammation/infection of myocardium and intramural vessels is detectable in patients with HIV-associated PAH. It may adversely affect right ventricular function and have a role in the compromised pulmonary circulation. Its recognition and treatment may influence progression of right ventricular dysfunction and perhaps PAH severity.
A.F., N.P., D.V. and C.C. contributed in the study's conception, design, and performance; M.F., R.B. and R.V. contributed in the collection and analysis of data; F.F. and G.I. contributed in writing and critical revision of the manuscript.
Source of funding: The study has been supported by the Grant RF-2009–1511346, by the Grant RBFR081CCS and by the Grant MRAR08Y012 from the Italian Ministry of Health and by the Grant ‘Ricerca Corrente’ INMI L. Spallanzani.
Conflicts of interest
Disclosure: No economic relationships with industry.
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