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How do we measure pathology in PAH (lung and RV) and what does it tell us about the disease Rubin M. Tuder Q1 Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado School of Medicine, 12700 East 18th Avenue, Research Complex 2, Room 9001, Aurora, CO 80045, USA

The current understanding of the pathology that underlies pulmonary vascular and right ventricular remodeling in pulmonary hypertension is discussed. Although recent studies underscored the importance of intima and media remodeling and, for the first time, the relevance of perivascular inflammation, much is needed to move the field forward. Reassessment of distribution and extension of the different vascular lesions requires state-of-the-art stereological tools, allied to three-dimensional casting and integration with data concerning cellular and molecular pathobiological processes. This integrated approach is ever more pressing in the right ventricle, because our understanding of key structural alterations of the failing right ventricle in pulmonary hypertension is lacking. This enterprise will enable better translation of pathogenetic processes to the human disease and provide key data to guide diagnostic and prognostic imaging approaches. Introduction More than 100 years since it was originally reported [1], pulmonary hypertension (PH) still lacks a comprehensive description regarding the type and extent of pulmonary vascular remodeling. Past pathological studies, summarized in [2], have been largely descriptive and of limited scope owing to methodological shortcomings; moreover, these studies did not consider the quantitative characteristics of the normal pulmonary arterial circulation. The anatomic and microscopic characteristics of the normal pulmonary circulation have recently been summarized in detail [3]. These insights were based on a handful of intravascular casting studies of human and rodent normal lungs, which annotated the branching pattern, starting at the smallest casted segment (i.e. branch 1) and Q2 increasing sequentially toward the hilar segments (i.e. branch 17). Despite limitations of this restricted approach (e.g. few samples, assumptions made to account for missing casted segments, etc.), several key findings can be appreciated. Approximately 108 arterial segments exist at the level of pre-capillary arteries in the range of 20 mm in diameter. Between the segments sized 200 mm to 20 mm in diameter there are approximately 2  106 segments. The ratio of surface area of the intima (which is proportional to the luminal E-mail address: [email protected].

area) to volume of the muscular (media) layer is the largest at precapillary level when compared with more-proximal pulmonary artery segments (i.e. hilar and segmental); this property enables low resistance and hence low blood perfusion pressure before it reaches the capillaries for gas exchange. It is also of interest that the largest arterial segments to contain a distinct muscular layer are in the range of 700 mm in diameter [4], suggesting that the control of vasotone could extend from the second-order of pulmonary arterial branching (in arteries of 40 mm in diameter – the smallest segment to contain a well-defined muscle layer) to the eighth-order, located in lung subsegments. Larger segments (greater than eighth-order) have an elastic-rich media, and probably contribute little to vasomotor regulation. Taken together, these data provide important anatomic landmarks to be considered when attempting to understand the scope of pulmonary vascular remodeling in PH. The persisting challenge in understanding pulmonary remodeling in PH is to define how the structural, cellular and molecular characteristics of the normal pulmonary circulation change in the disease setting. Because normal parameters were established based on the study of a small number of disease-free lungs, it is unclear whether the pulmonary circulation, before the development of PH in a given patient, had a different baseline structure when

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compared with individuals who do not develop the disease. Moreover, the molecular characteristics are probably not the same along the normal pulmonary artery segments; indeed, we noticed that smaller vascular segments (500 mm) of the normal pulmonary circulation [5]. As highlighted above, the regulation of blood flow could also depend on the geometric characteristics of the specific segments of pulmonary arteries, which, when coupled with a specific molecular program, can regulate tone and therefore affect pulmonary artery pressures in health and disease. There are numerous potential predictions regarding how pulmonary artery remodeling might be present in patients with the disease. In light that the overall surface area could be required to decrease by 80% by the time PH develops, a lower number of moresevere intima lesions compromising more-proximal segments (which are less numerous often by a log factor) would suffice to surpass the PH threshold. Lesions can be focal or multiple in each vascular segment. Moreover, they can reside at branching points, which can lead to compromise of blood flow on daughter segments with a single lesion. This particular outcome relies on specific predisposition of these vascular sites vis a vis a stochastic Q3 progression of vascular lesions within the up-to-17 pulmonary arterial segments. Notwithstanding these limitations, we have been able to analyze more in-depth a large number of lung samples with pulmonary arterial hypertension (PAH) [2], which provided us with several key findings and advanced our understanding of the spectrum of the pathology of the disease. The critical analysis of these data and prior reports on the pathology of PH reveal important remaining challenges that form the basis of this review.

Pulmonary vascular remodeling in PAH Instituted in 2006, the Pulmonary Hypertension Breakthrough Initiative (PHBI) consists of a North American initiative aimed at implementing a modern tissue bank of lungs with PAH. This Q4 large set of normal and PAH lungs allowed us the unique opportunity of determining the pattern of pulmonary vascular remodeling in the modern era of therapies targeted to this disease in a large set of lung tissues with PAH. The PHBI was launched under the sponsorship of the Cardiovascular Medical Research and Education Fund (CMREF) [2] (Pulmonary Hypertension Breakthrough Initiative: http://www.ipahresearch.org/PHBI-Research-Project.html). The sampling design used for collection of lung tissues was conceived with the goal of expediting the collection process (because tissues were also harvested for degradation-sensitive protein and mRNA isolation), while enabling a systematic approach in which the same regions of the right lung would be sampled; this design is described in detail in [2]. The major implication of this approach is that the data obtained from these tissues pertain to the regions that have been sampled; this approach entails an important limitation in that the data cannot be generalized to all regions of the lung or vascular segments present in regions of the lung that have not been sampled [3,6]. Nevertheless, several findings of our study are worth further discussion. These include the relevance of intima and media remodeling, the high degree of heterogeneity of pulmonary vascular lesions within a single lung and among PAH lungs, the lack of correlation between the number of profiles of plexiform lesions 2

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and hemodynamics and the potential impact of perivascular inflammation [2] (Fig. 1). Intima lesions are indeed the most characteristic morphological finding in severe PH. They involve proliferation of endothelial cells (forming the plexiform lesions), accumulation of myofibroblasts (intima obliteration or fibrosis) and accumulation of extracellular matrix including collagen and mucopolysaccharides [7,8] (Fig. 1). Often, intima remodeling leads to almost complete obliteration of the vascular lumen. Their exact location within the Q5 pulmonary arterial segments and overall distribution remain unclear. A single study based on 3D reconstruction of a small fragment of lung tissue identified that obliterative lesions in PH, associated with congenital heart malformations, occurred at branching points, often as isolated lesions [9]. We subsequently validated these observations when we undertook a similar goal to locate and establish the topographical relation of intima lesions using 3D reconstruction of vascular segments affected by iodiopathic PAH (IPAH) [10]. Moreover, the relation among these lesions (i.e. whether they follow a sequential pathobiological process) also remains unknown. Based on a rat model of severe PH due to the combination of chronic hypoxia and vascular endothelial growth factor (VEGF) receptor inhibition with SU5416 [11], we have speculated that the intima lesions could start and develop as a result of proliferation of endothelial cells forming plexiform lesions [12,13]. Furthermore, the heterogeneity of presentation of different forms of intima lesions not only within the same lung but also among individuals is noteworthy. There are patients whose lungs show numerous plexiform lesions, whereas others show a widespread predominance of obliterative lesions with few plexiform lesions. It is unclear whether these pathological phenotypes play any part in the clinical presentation or response to therapies. Although intima lesions were clearly more severe than intima remodeling seen in control lungs, the combination of intima and media remodeling had the most significant correlation with pulmonary artery pressures (it is noteworthy that all patients were listed for transplantation because of progressive and ‘terminal’ disease). Although muscular (i.e. media) remodeling also correlated with hemodynamic parameters, three of the four quartiles of media remodeling in PAH were equally shared with control lungs – only the fourth quartile with more extensive media thickness fell well above the thickness seen in control lungs. These findings give rise to several interesting considerations regarding the specific contribution of media remodeling to PAH. The extent of intima and media remodeling appears to progress independently because we could not find a close correlation between both forms of remodeling; however, media remodeling could antedate intima remodeling as suggested by the few human studies with sequential lung biopsies [14]. Intima remodeling could be linked to alterations of pulmonary artery flow imparted by media remodeling and vasoconstriction or, alternatively, develop as an independent event. In fact, both of these hypotheses have been partly supported by the sequential examination of the remodeling in the rat SU5416-chronic-hypoxia model [13]; this model is characterized by early media remodeling, reaching a plateau at approximately three weeks, when plexiform-like lesions develop, followed by progressive obliteration by complex vascular lesions. Although not stringently proven, inhibition of media

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Intima lesions: obliteration, concentric, plexiform

Media lesions: hypertrophy/proliferation

endothelial cells and myofibroblasts

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Pulmonary vascular remodeling pulmonary hypertension Perivascular inflammation: lymphocytes, mast cells, macrophages, monocytes (d)

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FIGURE 1

Q10 Summary of key pathological alterations in pulmonary arterial hypertension (PAH) lungs, involving intima (a, b) and media (c) remodeling and perivascular inflammation (d). (a) Obliterative intima lesions with fibrosis and acellular matrix (arrows). (b) Plexiform lesion (arrow). (c) Media hypertrophy (arrows). (d) Perivascular inflammation (arrows) surrounding a complex pulmonary vascular lesion (short, large arrows).

remodeling would probably prevent the development of the intima lesions, because triggers of media remodeling, like hypoxia, are required for the full development of severe PH in the model [11]. What are factors that account for the observed lesional heterogeneity seen in PAH lungs? One possibility is that different sampling approaches impart a significant error (or bias), inflating the differences among different regions of the same lung. Rather than relying on the assessment of profiles (i.e. outline of 3D objects in a 2D section of tissue), the correct approach would have required estimation of the number of lesions using a physical or optical dissector in a given sampled volume of lung tissue. Profiles are largely determined by the relative size of the lesion in question: smaller lesions, although possibly present even in a higher number, would be detected less frequently than more-sparse larger lesions [6]. If the aspect of heterogeneity would be confirmed with this state-of-the-art approach, then several interesting alternatives could be considered. Specific lesions would develop as a result of blood flow patterns and local disturbances, rather than following a prototypic sequence. Alternatively, lesions would be specified by the local geography of the arterial segments before the development of the lesions. Finally, the host’s ‘misrepair’ gene products

could also determine how lesions evolve and the speed and rate that they would evolve – a case in point would be the emergence of endothelial cell proliferation leading to the formation of plexiform lesions. Plexiform lesions, the characteristic lesion of PAH, have been the subject of intense debate regarding their pathobiologic relevance. They have been thought to represent terminal stage lesions [15], possibly related to suprasystemic pulmonary artery pressures [16]. The distribution of plexiform lesions in IPAH lungs remains unknown; as mentioned previously, the determination of the number of plexiform lesions has been biased because of the quantification of profiles – the estimated frequency of plexiform lesions rather represents a biased estimate based on size and a restricted sampling of pulmonary arteries [17,18]. Although limited by similar biases as those incurred by prior studies, we quantified the profiles of plexiform lesions [2]. The apparent advantages of our approach consisted of a large number of lungs in the PAH groups and a systematic sampling approach. We found a striking heterogeneity of occurrence of plexiform lesion in different regions of the same lung and among PAH lungs. Although plexiform lesions are only seen in severe forms of PH,

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notably in groups 1 and 2 PH we did not observe a significant correlation between the number of profiles and pulmonary hemodynamics. These findings do not invalidate the concept that obliterative intima lesions could involve angioproliferation in PAH [19,20]; it is conceivable that intima injury triggers endothelial cell proliferation, which can subsequently remodel to obliterative lesions (either fibrotic or concentric lesions). Once again, the SU5416-chronic-hypoxia model lends support that plexiform lesions could indeed represent the forerunner to obliterative lesions [13]. Adventitia remodeling failed to correlate significantly with hemodynamics in our cohort of PAH lungs. This is in contrast to an earlier extensive study of remodeling in IPAH [18]. This discrepancy can be explained by methodological differences and the difficulty in precisely defining adventitia boundaries. Our data do not detract from the growing importance of the adventitia niche in coordinating media remodeling and perivascular inflammation, a key process in PH.

Pulmonary artery inflammation in PAH Our studies were the first to establish the relevance of perivascular inflammation in PAH [2], because it correlated with pulmonary hemodynamics. Inflammation was recognized more than 20 years ago as an integral part of IPAH [21]. However, a persistent question in the field is whether inflammation drives the remodeling process or trails signaling triggered by altered blood flow and intravascular remodeling events. Furthermore, it is unclear whether the inflammatory process is nonspecific or rather directed against specific antigens. Specific immunity has been supported by the finding of autoantibodies in sclerodermaassociated PAH [22] and, more recently, by the presence of lymphoid follicles in IPAH lungs [23]. The types of inflammatory cells surrounding the remodeled pulmonary arteries, initially addressed with a handful of cell surface markers [24], have now been detailed with a more comprehensive immunophenotypic approach [25]. For instance, the finding of decreased numbers of Treg cells in IPAH lungs (when compared with normal lungs) further underscores the potential for an autoimmune-driven process in PAH. Macrophages and monocytes are key cells identified in all reports of inflammation in PAH [24,25]. The profile of macrophages increases from twofold to fivefold around pulmonary arteries ranging from 20 to >150 mm in diameter, mostly in the adventitia [25]. The macrophage influx can be seen as part of an innate response, triggered by factors such as hypoxia, cytokines or vascular injury and mediated by increased chemokine expression in the vicinity of pulmonary vascular lesions [26]. How can macrophages affect PH and pulmonary vascular remodeling? They can be a source of growth factors, such as platelet-derived growth factor (PDGF) [27] and transforming growth factor (TGF)-b [28]. Recent evidence also pointed to macrophages as sources of leukotriene B4 (LTB4), triggering endothelial cell death and smooth muscle cell proliferation [29]. A complementary role of macrophages would consist of directing adaptive immunity in the context of a TH2driven inflammation. PAH due to Schistosomiasis, the most frequent cause of PH worldwide, involves interleukin (IL)-13/TGF-bmediated inflammation and pulmonary vascular disease, possibly coordinated by alternatively activated macrophages [30]. 4

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An additional potential pathogenetic factor linking inflammation and pulmonary vascular remodeling involves alterations of bone marrow cells and the recruitment of bone marrow progenitors. Indeed, CD133/CD45-positive precursors have been demonstrated in histological sections of PAH lungs [31]; PAH patients have increased circulating progenitors [32], which can promote vascular injury when instilled in nude mice [33]. The overall pattern of inflammation and potential involvement of progenitor cells in the pulmonary vascular remodeling raises important considerations in relation to the use of progenitor or stem cells to treat the disease. Indeed, genetically modified circulating progenitors ameliorate experimental pulmonary hypertension [34]; mesenchymal stem cells, possibly via secretion of paracrine factors, can also attenuate pulmonary hypertension in the setting of bronchopulmonary dysplasia [35]. Given that PAH is a systemic disease, possibly involving the bone marrow [33], disease-specific progenitor cells could carry key phenotypic determinants that promote disease; by contrast, ‘disease-free’ progenitors or stem cells can be harnessed potentially to treat pulmonary hypertension.

Right heart remodeling in PAH The realization of the importance of failure of the right ventricle (RV) in PH has generated a growing interest in this area of investigation, because RV dysfunction is a critical determinant of morbidity and mortality in the disease [36]. Although there is abundant data available on the pathology of pulmonary vascular remodeling in PAH (despite the caveats discussed above), there is a vexing lack of data regarding microscopic, cellular and, notably, molecular features underlying the remodeling of the RV in PH. Given the lack of human data, a growing body of data has been generated using animal models, making it imperative to evaluate the quality of the available approaches and the ensuing conclusions of these studies, because the strength of this experimental data clearly impacts how they are translated to the human disease. There are some key structural components of RV remodeling: cardiac muscle hypertrophy, interstitial fibrosis and capillary density. Many of the approaches used to assess these parameters are similar to those used in the assessment of pulmonary vascular remodeling in PH. These involve planimetric measures, often reliant on digital imaging software (such as ImageJ, Metamorph1, Q6 Image-Pro1, etc.). What do these measurements mean? First and foremost, the sampling of the RV tissue suffers from the same caveats outlined to the analyses of lung tissue: they were done without a design or plan in mind, imparting an important bias. This means that the data cannot be extrapolated to the entire RV tissue in a given experimental sample or to a sample of individuals that is representative of an experimental population. Most importantly, these caveats do not allow comparisons across different studies. The planimetric approaches are biased toward profiles – the larger the individual parameters the higher the chance that they will be ‘measured’ by these approaches. An integral component of RV remodeling is myocyte hypertrophy. This means that the total volume of myocytes increases in RV remodeling as the myocytes become ‘bigger’. The most frequently used measure for myocyte hypertrophy is the determination of cross-sectional area, which can be affected by biased sampling. We propose instead the use of validated stereological approaches [6],

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FIGURE 2

Summary of processing steps for right ventricle (RV) stereology. (a) RV and left ventricle (LV) of a rat heart are dissected free from the atria (1); RV is dissected from septum and LV (2); RV is weighed (3); RV volume is documented by water displacement (4) [44]. RV is embedded in molten paraffin (5), which is then placed in spherical molds to form paraffin balls (or isector) (6); to randomize in three dimensions; the isector is allowed to rotate and the point of contact with the surface used to define an unbiased sectioning place (8); parallel slices are taken from the RV (generally every 3 mm) using a slicing mold (9); the RV slices (n = 4) are then re-embedded with the specific sectioning place established by the isector (10) and unbiased histological sections performed (11). The isector allows unbiased estimates of orientationdependent stereological measures involving surface area and length. (b) Example of a rat RV section with capillaries stained with Griffonia simplicifolia lectin (red) and cardia-specific myosin (green). The free program STEPanizerß (http://www.stepanizer.com/) was used to quantify the number of intersects of capillaries in the counting plane (framed within solid and dashed lines), whereas the counting grid (stars) was used to determine interstitial and myocyte volumes. www.drugdiscoverytoday.com 5 Please cite this article in press as: Tuder, R.M. How do we measure pathology in PAH (lung and RV) and what does it tell us about the disease, Drug Discov Today (2014), http://dx.doi.org/ 10.1016/j.drudis.2014.05.022

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which provide the absolute volume of the RV myocytes, relying on determining heart volume (either by the Cavalieri’s method or random stratified sampling) [37] and then the volume density of RV myocyte to RV (by point counting). The same approach would enable the determination of the total volume of interstitial fibrosis, often highlighted in animal models of heart failure and PH based on profile measurements (Fig. 2a). Assessments of the RV capillary network have not applied stereological rules [38,39]. These studies reported capillaries as being reduced, based on a decrease in ‘capillary density’, often determined using digital planimetric assessment. Capillaries, like dendrites or axons, require estimates of length, which is orientation dependent (remember how the length of a pencil is dependent on how you hold it in three dimensions). This requirement can only be fulfilled by randomizing the histological sections in three dimensions – like using the isector [40]. Furthermore, bidimensional representation of lines (which appear as points) requires the superposition of a plane-counting tool (also known as probe), because the number of times that a line traverses a plane provides a measure of its length [37] (Fig. 2). Conceptually, the efficiency of oxygen supply will be determined by the capillary length, the interface between the red cells and the target cell, the cardiac myocyte and the volume of the myocytes. It is noteworthy that studies that verified a ‘loss of capillaries’ did not comply with these requirements [38,39]; moreover, they have not addressed whether there is tissue hypoxia using the hypoxia probe pimonidazole [41]. Capillaries are sometimes difficult to identify in plain histological sections, often requiring their staining with antiboQ7 dies (line factor-VIII-related antigen or CD31); this approach introduces another bias regarding the requirement of obligatory reactivity with the antibodies – lack of staining could reflect loss of antigen expression rather than true loss of capillaries. We have determined that the lectin Griffonia simplicifolia provides the most reproducible highlight of capillaries with no apparent loss of signal in remodeled RV when compared with normal RV (Fig. 2b). Moreover, confocal imaging, which enables delineation of capillary volume in three dimensions, suffers from biases imposed by the restricted sampling of the tissue. These stereological principles and approaches are key for measuring other relevant processes in the failing and normal RV. These include cardiac myocyte and nonmyocyte proliferation or apoptosis, which can explain the remodeling process and RV dysfunction in PAH. In summary, the structural elements that underlie RV hypertrophy and failure, particularly in human PAH, remain to be analyzed with state-of-the-art stereological approaches. These approaches

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have been recently reviewed [42], and they can be instrumental in characterizing RV remodeling in humans and animal models.

Concluding remarks It is apparent that the knowledge in the pulmonary vascular field, particularly in PH, has progressed immensely since its early inception. The most important landmark achievement in the field was the introduction of pulmonary catheterization [43], which defines the disease up to the present date. Despite the agreement that PH involves and is possibly driven by pulmonary vascular lesions, it is apparent that we lack the means to image the altered pulmonary circulation, a key step to advance PH research and clinical care. Because imaging modalities remain to be developed, it is pressing that we further our understanding not only on the molecular and cellular nature of PH but also regarding how the lesions are distributed and the extent of the vascular compromise in PH lungs. The availability of resected PAH lungs via the PHBI and other initiatives creates a unique opportunity that will certainly have a long-standing impact toward the goal of ultimately imaging pulmonary vascular lesions. The limitations of most if not all descriptive studies of pulmonary vascular remodeling in PH could have contributed to slowing the translation of pathogenetic insights into clinical management of patients with the disease. Because we lack insights into the precise distribution and extent of pulmonary vascular lesions (at the anatomical level using explanted lungs and in patients as a result of the inability to image the pulmonary circulation), evaluation regarding efficacy of therapies cannot be properly assessed. These have been replaced by surrogate markers like time to clinical deterioration or the 6-minute walk test. Importantly, validation of potential pathogenetic mechanisms might have been curtailed by the limitation of the descriptive methods used thus far [3]. The present review attempts to balance between our current understanding of PH pathology and the realization of methodological limitations underscoring this body of knowledge. We hope that the critical review of these data might serve as a template for more-definitive future studies. These are crucially needed as a required framework for the development of diagnostic and prognostic imaging tools. Their availability will certainly impact the field decisively, very much like that followed by the introduction of pulmonary hemodynamic measurements to characterize PH.

Acknowledgment Supported by the Cardiovascular Medical and Research Educational Fund (to R.M.T.).

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www.drugdiscoverytoday.com 7 Please cite this article in press as: Tuder, R.M. How do we measure pathology in PAH (lung and RV) and what does it tell us about the disease, Drug Discov Today (2014), http://dx.doi.org/ 10.1016/j.drudis.2014.05.022

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Drug Discovery Today  Volume 00, Number 00  June 2014

How do we measure pathology in PAH (lung and RV) and what does it tell us about the disease.

The current understanding of the pathology that underlies pulmonary vascular and right ventricular remodeling in pulmonary hypertension is discussed. ...
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