Three linked vasculopathic processes characterize Kawasaki disease: a light and transmission electron microscopic study

Abstract

Background: Kawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course.

Methodology/principal findings: Specimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an "eosinophilic-type" myocarditis.

Conclusions/significance: NA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.

Figure 1. Kawasaki Disease Vasculopathy, Process 1, Necrotizing Arteritis (NA).

NA is an acute self-limited, one-time, synchronous process complete within about 2 weeks of fever. It starts at the endothelium of medium sized muscular and elastic arteries and progresses peripherally; involvement of veins, pulmonary arteries, and aorta is not observed.

Figure 2. Necrotizing Arteritis (NA) and severe SA/C pan-arteritis.

A. Portion of a CA undergoing NA. The friable fragmenting wall is a mixture of neutrophils and debris. The adventitia is virtually obscured by inflammation and RBCs. H&E, case 2, original magnification 16×. B. Higher magnification of an area of CA NA predominantly of neutrophils. H&E, case 2, original magnification 63×. C. The necrotizing process has reached into the adventitia, which is only mildly inflamed. H&E, case 2, original magnification 16×. D. An area of CAA consistent with having undergone severe SA/C pan-arteritis, leaving only adventitia rich in SA/C inflammatory cells, almost exclusively small lymphocytes. Note the longitudinally sectioned vessel and fibrin/RBC lining the luminal surface. H&E, case 11, original magnification 40×. L=lumen, ADV=adventitia, V=vessel, F=fibrin, NA=necrotizing arteritis, N=neutrophils.

Figure 3. Kawasaki Disease Vasculopathy, Process 2: Subacute/Chronic (SA/C) Vasculitis.

A ubiquitous, asynchronous inflammatory process that can begin as early as the first two weeks, and targets medium-sized muscular and elastic arteries. Inflammatory cells: small lymphocytes>>eosinophils & plasma cells>> macrophages. Minimal (subclinical) involvement of veins, pulmonary arteries, & aorta. Triggers Process 3, luminal myofibroblastic proliferation (LMP).

Figure 4. Kawasaki Disease Vasculopathy, Process 3: Luminal Myofibroblastic Proliferation (LMP).

A process triggered locally and apparently remotely by Process 2 that can progress to total occlusion. LMP myofibroblasts are ultrastructurally, but not functionally, similar to wound healing myofibroblasts.

Figure 5. Subacute/Chronic (SA/C) Inflammation in KD CAA.

A. SA/C inflammatory cells extending from the adventitia of a CA has almost reached the IEL. The EEL and most of the media are obscured/destroyed by the SA/C. The lumen contains a mixture of RBC, fibrin, and lysing leukocytes, that could indicate the beginning of a clot. H&E, case 11, original magnification 40×. B. The CA adventitia and media are rich in SA/C inflammatory cells. The thick inflammation of SA/C-LMP has obscured most of the myofibroblasts. There is more collagen staining (blue) in the adventitia than protein staining of the ECM of the SA/C-LMP. Note the increase in cellular concentration toward the lumen. Trichrome stain, case 18, original magnification 16×. C. The alpha-SMA stains SMC and vessels in this CA adventitia, SMC in the media, and MF in the SA/C-LMP. Focal areas of IEL remain. Alpha-smooth muscle actin (SMA) immunohistochemistry (IHC), case 18, original magnification 10×. D. At higher magnification, SMCs appear to be entering the media from the adventitia, while large myofibroblasts exit the luminal side into the SA/C-LMP. SA/C inflammation is present in all three layers. Alpha-SMA IHC, case 18, original magnification 63×. M=media, SMC=smooth muscle cells, MF=myofibroblast, ADV=adventitia, IEL=internal elastic lamina, LMP=luminal myofibroblastic proliferation, L=lumen, V=vessel, P=pericardium.

Figure 6. Luminal Myofibroblastic Proliferation (LMP) in KD CAA.

A. The peri-luminal portion of the CA SA/C-LMP is rich in polygonal shaped myofibroblasts, while elsewhere the myofibroblasts are more pleomorphic. The background is mostly small lymphocytes. Alpha-SMA IHC, case 18, original magnification 63×. B. At higher magnification, the myofibroblasts in this area of SA/C-LMP resemble a culture of pleomorphic mesenchymal cells. The SA/C background is especially rich in small lymphocytes. Alpha-SMA IHC, case 13, original magnification 100×. C. The LMP resembles a “syncytium” of pleomorphic MF with red-staining actin and blue-staining pro-collagen. SA/C nuclei stain dark red. There is some blue staining of the ECM. The luminal lining (top) consists of RBCs and dark red staining fibrin. Trichrome stain, case 13, original magnification 100×. D. The SA/C-LMP contains large, pleomorphic myofibroblasts with large nuclei and several small nucleoli. The ECM is difficult to delineate. Trichrome stained plastic section, case 13, original magnification 160×. L=lumen, MF=myofibroblast, LMP=luminal myofibroblastic proliferation.

Figure 7. SA/C-Luminal Myofibroblastic Proliferation (LMP) causes narrowing of CA in KD patients.

A. A tear drop-shaped CA with SA/C-LMP is about 50% narrowed. The actin-rich myofibroblasts and medial and adventitial SMCs stain brown. The residual media is variably thinned. Alpha-SMA IHC, case 11, original magnification 10×. B. Oval-shaped SA/C-LMP CA is virtually occluded. The MF and medial SMC stain red and there is a relatively little ECM (blue) in the LMP. Except for some thinning, the media is almost completely intact. The adventitia is densely collagenized. Trichrome stain, case 28, original magnification 10×. C. The SA/C-LMP in this elongated CA is eccentric having started at the end (top) where the media and IEL had been destroyed and progressed toward the end, which still contains media and IEL (bottom). H&E, case 26, original magnification 10×. D. An oval CA is occluded by SA/C-LMP, of relatively low SA/C cellularity concentrated around the lumen. There are several small areas of minimally preserved media. No media is present on the side with the hyper-vascular adventitia, where the process likely began. H&E, case 27, original magnification 10×. E. The SA/C-LMP progressed from the inflamed/damaged end (bottom) of this tangentially-sectioned renal artery almost reaching the opposite end (top), where the lumen is visible and there is intact media and IEL are intact. H&E, case 27, original magnification 10×. L=lumen, M=media, ADV=adventitia, LMP=luminal myofibroblastic proliferation, RBC=red blood cells, IEL=internal elastic lamina, V=vein, I=inflammation, T=renal tubules, P=pericardium, My=myocardium.

Figure 8. Thrombi in coronary artery aneurysms (CAA) of KD patients.

A. A CA already severely compromised by SA/C-LMP is virtually occluded by a superimposed fresh thrombus (dark red) that blends into the SA/C-LMP. Trichrome stain, case 18, original magnification 16×. B. Part of a NA CAA occluded by a fresh thrombus. There is a small area of organizing thrombus. Since only mildly inflamed fibrotic adventitia remains, it is not possible in this section to distinguish between NA and SA/C as the etiology. H&E, case 11, original magnification 10×. C. A small portion of CAA thrombus. The vascular granulation tissue has a loose matrix containing few free RBC, spindle cells and mononuclear cells, a few of which are macrophages containing brown hemosiderin blood pigment. Spindle cells are reaching into the luminal RBCs. H&E, case 22, original magnification 63×. D. The oldest peripheral thrombus in this CAA is re-canalizing. There are superimposed fresher thrombi. Since the CAA still has some remaining media, the aneurysm likely resulted from severe SA/C pan-arteritis. H&E, case 37, original magnification 10×. E. This organized SA/C CAA thrombus has peripheral clumps of calcium. Some of the media is still visible (upper right). H&E, case 19, original magnification 16×. L=lumen, LMP=luminal myofibroblastic proliferation, M=media, ADV=adventitia, ORG=organizing thrombi, R=recanalized, Ca=calcium, V=vessel.

Figure 9. Myocarditis.

A. Low magnification H&E of a poorly-preserved hourglass-shaped epicardial CA showing varying degrees of SA/C peri-arteritis, transmural SA/C, and minimal preserved media. H&E, case 1, original magnification 10×. B. Perivascular and transmural SA/C inflamed adventitia and heavily damaged media with some discernible SMC and IEL, and SA/C-LMP with scattered pleomorphic myofibroblasts. There are some eosinophils intermixed with the small lymphocytes. H&E, case 1, original magnification 16×. C. A higher magnification of a typical area of SA/C-LMP with prominent amphophilic myofibroblasts in a background of mostly small lymphocytes, scattered eosinophils, and likely macrophages in a fibrillar ECM that shows some artifactual spaces. H&E, case 1, original magnification 25×. D. A typical area of the highly edematous interstitial myocarditis especially rich in eosinophils, with scattered lymphocytes, macrophages, and plasma cells. Note the longitudinally-sectioned caterpillar-shaped and cross-sectioned, owl eye-shaped (unidentified) Anitschkow chromatin pattern in a myocyte and two unidentified cell nuclei, respectively. H&E, case 1, original magnification 40×. L=lumen, NV=nerve, IEL=internal elastic lamina, Eo=eosinophil, AM=Anitschkow myocyte, MF=myofibroblast, V=vein, ADV=adventitia, M=media.

Figure 10. Early transitional changes of medial SMC into myofibroblasts.

A) Tangential section of media showing variably oriented electron dense banded collagen (e.g., C). The SMC are somewhat swollen, yet the actin (A)/dense bodies (long red arrows) are still apparent. The external lamina is obscured by the ECM. The dense plaques (short black arrows) are visible. Case 32, original magnification 5,000×. B) Longitudinal section of medial SMCs commencing their transition to MF. There is abundant actin (A) with dense bodies (long red arrows), dense plaques (short black arrows), shedding external lamina (short red arrows), and serrated nuclei. Electron dense banded collagen production has increased, and there is fibronectin (long black arrows). Higher magnification showed abundant pinocytic vesicles. Case 32, original magnification 4,000×.

Figure 11. Increase in myofibroblast RER and pleomorphism, as well as intercellular elastin.

A) The actin (A)/dense bodies (long red arrows) share the cytoplasm with RER. The extracellular matrix is composed almost exclusively of haphazardly arranged electron dense banded collagen. The ECM obscures the dense plaques and external lamina. Pinocytic vesicles (P) are visible. Case 32, original magnification 5,000×. B) Dense plaques (short black arrows), actin (A), and dense bodies (long red arrows) have decreased and there is more RER. Note the abundant electron dense intercellular elastin (E), mixed with the collagen. Note the external lamina (long black arrows). Case 32, original magnification 5,000×.

Figure 12. Myofibroblast fibronectin, shed external lamina, and intercellular junction.

A) There is copious actin (A) with prominent dense bodies (long red arrows) in this large pleomorphic myofibroblast. The nucleus is also large and irregular. Note the electron dense fibronectin (long black arrows). The ECM is loose and contains shed external lamina (short red arrows). Case 32, original magnification 5,000×. B) The cell has two prominent visible cytoplasmic extensions and is shedding strips of external lamina (short red arrows) into a loose stroma of granular-fibrillar ECM, with no banded collagen. Dense bodies (long red arrow), actin (A) and RER are present within the cell. Case 26, original magnification 5,000×. Inset: A non-specific junction (JXT) joins two MF. Actin (A) is present with very prominent dense bodies (long red arrows). Case 13, original magnification 10,000×.

Figure 13. Copious cytoplasmic RER and shed external lamina and fibronectin.

A) The slightly tangentially sectioned cell has about equal RER and actin (A) with dense bodies (long red arrows). There is a single large primary lysosome (*), shed external lamina (short red arrows), and relatively sparse dense plaques (short black arrows). Case 13, original magnification 5,000×. B) A MF with more RER than peripheral actin (A)/dense bodies (long red arrows). The nucleus is large and irregular with a prominent nucleolus and apparently lost some chromatin during processing. Shed external lamina (short red arrow) and fibronectin (long black arrow) are prominent. Case 13, original magnification 4,000×.

Figure 14. Pleomorphic mono- and binucleated myofibroblasts, abundant RER and SA/C lymphocytes.

A) RER dominates the cytoplasm of this stellate myofibroblast with a complex nucleus and at least two small nucleoli; some actin (A) is present. The matrix contains some shed external lamina (short red arrows) and fine filamentous collagen. There are typical small lymphocytes (L) with high nuclear to cytoplasmic ratios. Case 13, original magnification 1,700×. B) This MF is bi-nucleated and has very dilated, complicated profiles of RER plus actin (A). The loose ECM is dominated by shed external lamina (short red arrows). Case 26, original magnification 5,000×.

Figure 15. Explanted heart (transplant 2) with post-transplant CA vasculopathy showing similar features to those in KD LMP.

In patient 30, the second transplanted heart was removed because of post-transplant vasculopathy; pathologic myofibroblasts were observed in the luminal lesions of the coronary arteries. This myofibroblast is similar to the one in Figure 10B with its broad irregular processes (right field). It has extensive dense plaques (short black arrows), abundant actin (A) and some dense bodies (long red arrows). External lamina is both attached and shed (short red arrows). The loose banded collagen (C) is in the upper left of the field. Case 30C, original magnification 4,000×.