Hela l-CaD is Implicated in the Migration of Endothelial Cells/Endothelial Progenitor Cells in Human Neoplasms

Abstract

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. No functional differences among the isoforms in in vitro studies were found so far. In a previous study we found that the low molecular caldesmon isoform (Hela l-CaD) is expressed in endothelial cells (ECs)/endothelial progenitor cells (EPCs) in tumor vasculature of various human tumors. Activation of cell motility is necessary for the navigation of the tip ECs during angiogenesis, and migration of EPCs from the bone marrow during vasculogenesis. In the present study we searched for features of motility and the intracellular expression sites of Hela l-CaD in ECs/EPCs of various human tumors under histologically preserved microenviroment. We discovered a variety of motility-related cell protrusions like filopodia, microspikes, lamellipodia, podosomes, membrane blebs and membrane ruffles in the activated ECs/EPCs. Hela l-CaD appeared to be invariably expressed in the subregions of these cell protrusions. The findings suggest that Hela l-CaD is implicated in the migration of ECs/EPC in human neoplasms where they contribute to tumor vasculogenesis and angiogenesis.

Introduction

Caldesmon (CaD) is a major actin cytoskeleton (CSK)-associated protein that serves as a target for a variety of signalling pathways which modulate cell contractility, motility, division and adhesion in fully differentiated smooth muscle cells (h-CaD, 120–150 kDa) and in various other cell types (l-CaD, 70–80 kDa).^1,2 The exon 1-containing splicing variants and protein isoforms of l-CaD were initially cloned from Hela S3 cell lines and subdivided into Hela l-CaD I and II.² In quiescent cells l-CaD is associated with actin filaments (stress fibres) and tropomyosin (TM).¹ Stress fibres terminate at focal adhesions where l-CaD, TM and myosin are not present.³ The associations between these proteins change during cell activation and mitosis by specific receptor ligand interactions.¹ In a previous study we found that in glioma vasculature the exon 1-containing transcripts of Hela l-CaD are abnormally spliced.⁴ This results in the altered expression of the protein isoforms in the endothelial cells (ECs)/endothelial progenitor cells (EPCs) which take part in neo-angiogenesis in various human tumor types.^4,5 We also found that abnormal splicing results in the upregulation of the entire l-CaD class in glioma tissue samples⁴ and body fluids.^6,7 We assumed that the abnormal expression of Hela l-CaD in neoplastic vasculature is implicated in tumor neovascularization.

In the normal situation, endothelial cells (ECs)/endothelial progenitor cells (EPCs) are quiescent. EPCs largely reside in the adult bone marrow and the fraction of circulating EPCs in the blood of healthy people is low.⁸ The cells become activated by environmental stimuli induced by physiological and pathological signals during vasculogenesis and angiogenesis upon which they proliferate and migrate.⁹ Upon activation of ECs/EPCs changes in focal adhesions occur and simultaneous remodelling of F-actin causes changes in the shapes of the cells.¹⁰ Actin-associated proteins play important roles in the physiology and pathology of the ECs/EPCs.^10,11 So far, little attention has been drawn to the role of CSK-associated proteins in ECs/EPCs taking part in tumor neovascularization. Activation of cell motility is a crucial event in the navigation of tip ECs during angiogenesis and the recruitment of circulating EPCs from bone marrow for vasculogenesis. The functional expression of CaD has been well described in cultured cells but has not been investigated in human tissue or tumors. We hypothesized that Hela l-CaD is implicated in cell migration. To this aim, we studied ECs/EPCs taking part in neoangiogenesis in various human tumors versus normal tissues by using confocal laser scanning microscopy (CLSM) on multiple-labelled slides. Motility-related cellular structures were identified and characteristic protein expression patterns in these structures are described in detail.

Materials and Methods

Frozen tissue samples.

Frozen tissue sections of five gliomas, three breast cancers, two renal cell carcinomas, and six tissue type-matched controls (two brains, two breasts and two kidneys), taken from the tissue bank of the Department of Pathology of the Erasmus Medical Center, Rotterdam, were used. The Ethical Review Board at the Erasmus Medical Center approved the use of human tissue samples.

Antibodies and relevant reagents.

The antibody (CUK-0129) specifically against the Hela-type isoforms was generated as described before.⁵ A panel of primary antibodies, including CUK-0129, CD133 (Abgent), KDR (Lab Vision), CD34 (Lab Vision), vinculin (Serotec), talin (Serotec), gelsolin (Sigma), smooth muscle actin (SMA) (Abcam), caldesmon, LMW (Lab Vision), tropomyosin (clone TM311) (Sigma), anti-phospho-Ser-789 (Epitomics, Inc), cortactin (Upstate Biotechnologies), anti-Arp2/3 (C-13) (Santa Cruz Biotechnology) were used. Species-mismatched secondary antibodies, including FITC-conjugated goat-anti-mouse (Southern Biotech), Cy5-conjugated donkey anti-rabbit (JacksonImmuno Research) and TRITC-conjugated sheep anti-mouse (Chemicon, Internal Inc), were used for the detection. Other relevant reagents used included TRITC-phalloidin (Sigma), 4′,6-Diamidino-2-phenylindole (DAPI)(Vector Laboratories) and CSA kit (Dako). Appropriate fluorescent-labelled isotype antibodies served as negative controls.

Immunofluorescence staining and image processing.

The slides were processed for immunofluorescence (IF) multiple labelling as previously described.⁵ For simultaneous detection of two antigens by the antibodies raised in the same species, we used the protocol as provided by the CSA kit.¹² CD34 expression is retained by most of the resting and activated ECs in adults¹³ or EPCs.¹⁴ Vascular endothelial growth factor receptor-2 (VEGFR-2) or kinase insert domain receptor (KDR) is a marker of hemangioblasts and is exclusively expressed in endothelial and hematopoietic stem cells.^15,16 CD133 is well known as a marker for progenitor cells.¹⁴ Thus, EPCs can be defined as CD34⁺/CD133⁺ or CD34⁺/KDR⁺¹⁷ or CD34⁺/CD133⁺/KDR⁺.¹⁴ Hela l-CaD-positive EPC were defined as CD34⁺/CD133⁺/CUK-0129⁺, or CD34⁺/KDR⁺/CUK-0129⁺. Vascular ECs are defined as CD34⁺/CD133^-/CUK-0129⁺ or CD34⁺/KDR^-/CUK-0129⁺. ECs/EPCs are defined as either CD34⁺/CUK-0129⁺ or KDR⁺/CUK-0129⁺. Antibodies against vinculin and talin are used for the visualization of their reorganization into podosomes. Antibodies against gelsolin, Arp2/3 and cortactin are used as markers for podosomes.^18,19 Anti-phospho-Ser-789 is used for the detection of phosphorylated CaD. Each phosphorylation site is uniformly conserved in all of the isoforms of CaD.²⁰ TRITC-phalloidin is used for the visualization of F-actin. SMA staining defines mural cells (smooth muscle cells/pericytes) in vessel walls. DAPI was used for nuclear staining. All IF labeling was examined by confocal laser scanning microscopy (CLSM) as described previously.⁵ Briefly, CLSM was performed using a Zeiss LSM 510 META system (Karl Zeiss, Gottingen, Germany). The laser diode 405 nm, argon 488 nm and HeNe 543 or 633 were switched on and set at 50% output intensity. We used a korr C-Apochromat 63 objective to generate a single image for each sample. DAPI, FITC, Cy3/TRITC or Cy5 preparations were scanned by using changes of the three or quadruple filters. The three or four signals were detected by the same detector and stored separately in the computer memory.

Quantification of CD34⁺/CD133⁺/CUK-0129⁺EPCs in the specimens.

CD133⁺ EPCs represent the most primitive subset of EPCs,²¹ which are only present in bone marrow²² or spleen.²³ We examined all control and tumor samples for the presence of CD133⁺ EPCs. Sections were scanned under CLSM for the simultaneous detection of signals derived from CD34, CD133, Hela l-CaD and DAPI. The frame size was set at 512 x 512. In each sample, 15 non-overlapping areas were scanned.

Estimating relative percentages of Hela l-CaD-containing ECs/EPCs with cellular protrusions in the specimens.

All protrusions searched for in this study are, except for the leading lamellae (visible under low magnification), only recognizable under very high magnification. Since the leading lamellae invariably appear together with other protrusions like lamellipodia, filopodia, microspikes, membrane blebs and ruffles, this type of protrusion (viz., the leading lamella) was used for quantification of protrusion-bearing EPCs/ECs in the specimens under low magnification.

Results

Characterization of subsets of EPCs and Hela l-CaD⁺ EPCs/ECs in tumor vasculature.

We did not detect CD133⁺ EPCs in any of the normal control samples. In the tumor samples three CD34⁺-based subsets of EPCs were identified by using combined immunostaining for CD133, CD34 and KDR: CD34⁺/CD133⁺/KDR⁺ cells (Fig. 1A); CD34⁺/CD133⁺/KDR^- and CD34⁺/CD133^-/KDR⁺ (Fig. 1B). Hela l-CaD ECs/EPCs are often present as multi-nucleated cells (Fig. 1C and D). CD34, KDR and CD133 not only have extracellular and transmembrane domains but also cytoplasmic domains (essential functional domains).^24–25 Therefore, these molecules may colocalize with cytoplasmic proteins in the cell images.

Figure 1.

Characterization of Hela l-CaD⁺ EPCs/ECs in tumor vasculature. (A) CD34⁺/KDR⁺/CD133⁺ EPCs are identified in a glioblastoma. (B) CD34⁺/KDR^-/CD133⁺ and CD34⁺/KDR⁺/CD133^- EPCs are detected in a breast carcinoma. (C ) Hela l-CaD is expressed in a multinucleated EPC in an anaplastic oligodendroglioma. (D) Hela l-CaD is expressed in a multinucleated EC (negative KDR staining) in a renal cell carcinoma. Scale bars = 20 µm.

Estimating percentages of CD34⁺/CD133⁺/CUK-0129⁺EPCs in different tumor types.

The CD34⁺/CD133⁺/CUK-0129⁺ EPCs are found individually or alternatively, form cell chains and clusters, or are incorporated into vascular walls (Fig. 2). The percentage of EPCs was calculated as the sum of the areas with the EPCs versus the total scanned areas (15) with normalization by 100. The percentages ranged from 27% to 93% (Table 1).

Figure 2.

CD34⁺/CD133⁺/CUK-0129⁺EPCs in various tumor types. (A) CD133⁺ EPCs are individually deposited in an anaplastic oligodendroglioma. Some of these cells form leading lamellae (arrows). (B) CD133⁺ EPCs are demonstrated as cell clusters within a solid vascular plexus in an invasive ductal carcinoma. (C) EPCs incorporated in the vessel walls in a renal cell carcinoma.

Table 1.

Percentage of CD34⁺/CD133⁺/CUK-0129⁺ EPCs in various tumors

Case	Organ	Tumor Type	Positive Areas	Percentage
1	Brain	Anaplastic oligodendroglioma	13/15	87%
2	Brain	Anaplastic oligodendroglioma	14/15	93%
3	Brain	Anaplastic oligodendroglioma	12/15	80%
4	Brain	Glioblastoma	11/15	73%
5	Brain	Glioblastoma	10/15	67%
6	Breast	Invasive ductal carcinoma	8/15	53%
7	Breast	Invasive ductal carcinoma	7/15	46%
8	Breast	Invasive ductal carcinoma	6/15	40%
9	Kidney	Renal cell carcinoma	5/15	33%
10	Kidney	Renal cell carcinoma	4/15	27%

Relative percentage of Hela l-CaD-containing ECs/EPCs with cellular protrusions.

Under low magnification, we screened 200 Hela l-CaD-positive ECs/EPCs and counted the number of cells with leading lamellae in each specimen. The percentage of Hela l-CaD-positive ECs/EPCs with leading lamellae ranged from 10% to 50%.

Asymmetric cell protrusions identified in ECs/EPCs of tumor vasculature.

Generally, quiescent cells are symmetric, but migrating cells are characterized by various asymmetric cell protrusions.²⁶ Since normal (quiescent) vascular ECs do not express Hela l-CaD as verified in our previous study,^4,5 we used general anti-l-CaD, recognizing all isoforms together with CD34, to define the overall symmetry of the cells which were captured from the luminal lining of vessels of normal tissues (Fig. 3A). None of cell protrusions were detected in normal (quiescent, Hela l-CaD negative) vascular ECs. A variety of cell protrusions characteristic of motile cells were detected in the Hela l-CaD⁺ ECs/EPCs in the tumor vasculature from the different tumor samples. Sheet-like regions of the cytoplasm to one side of the nucleus were interpreted as leading lamellae²⁷ (Fig. 3B–E). These protrusions change the overall cell contour. A lamellipodium is defined as a thinner, planar extension of the front of a leading lamella.²⁸ Lamellipodia vary in width from ∼1 to 5 µm and exhibit variable numbers of radiating microfilament bundles.²⁸ The proximal part of the bundle is usually embedded in the lamellipodial network and is designated as “nonprotruding filopodia” or “microspike”.²⁹ The distal part of the bundle protruding beyond the leading edge of the lamellipodia is designated as “filopodia”.²⁶ Hela l-CaD is bundled in microspikes and filopodia (Fig. 4A). The bundles are consistently present as monitored in a series of optical sections (z-stack) (Fig. 4B). Structurally, podosomes comprise of a core of F-actin and actin-associated proteins like Arp2/3 complex, gelsolin, cortactin and caldesmon, etc., embedded in a ring structure of other proteins.^3,30 Adhesion mediators such as vinculin or talin preferentially associate with the ring structure.^31,32 In this study, we detected localized clusters of F-actin, Hela l-CaD, TM, vinculin, talin, gelsolin, cortactin and Arp2/3, indicative of the presence of podosomes. The expression pattern of Hela l-CaD parallels that of F-actin. This molecule is present as punctuated or dot-like structures (Fig. 5A and B) in tumor ECs/EPCs as F-actin appears (Fig. 5C–I) but not in normal vascular ECs. Hela l-CaD and fragmented F-actin is preferentially localized in the “core domains of podosomes” (Fig. 5B, D, F, H and I). We also observed belt-shaped podosomes at the cell periphery (Fig. 5C) as described elsewhere.^31,33 Vinculin and talin are preferentially expressed around the cores of podosomes (Figs. 5B, D and F), while gelsolin i s predominantly present in these cores (Fig. 5H). The expression of cortactin and Arp2/3 is similar to that of gelsolin (data not shown). The expression of TM predominantly surrounds the core domains of podosomes (Fig. 5I). Gelsolin is well-characterized as marker for motile cells or serves as a capping protein.³⁴ In this study, gelsolin is not detected in normal vascular ECs (Fig. 6A), which is confirmed by double labelling of gelsolin and either CD34 (Fig. 6B) or SMA (Fig. 6C). The spherical membrane protrusions called “blebs”³⁵ in which Hela l-CaD was accumulated were observed either at the edge of lamellipodia (Fig. 7) or at the surface of ruffles (Fig. 8) in asymmetric protrusive cells. Membrane ruffles (Fig. 8) are interpreted as inefficient lamellipodial protrusions,³⁶ consisting of irregular protrusions of the margins of the cell surface membranes.³⁷ The expression pattern of phosphorylated CaD(pCaD) appeared to be homologous with that of Hela l-CaD (data not shown) in this study.

Figure 3.

Various patterns of leading lamellae observed in EPCs/ECs in tumor vasculature, but not in normal vascular ECs. (A) l-CaD and CD34 show a regular distribution in a vascular EC captured from a normal vessel. No protrusions are detected. (B) Crescent-like cytoplasmic protrusion of an EPC in a glioblastoma. (C) Irregular cytoplasmic protrusion of an EPC in an anaplastic oligodendroglioma (D) Broad-sheet-like protrusion of an EPC in breast cancer. (E) Long cytoplasmic protrusion of an EC in a renal cell carcinoma (negative KDR staining). Scale bars = 20 µm.

Figure 4.

Lamellipodium and microspike/filopodia in an EPC in tumor vasculature. (A) The cell is scanned at the z-axis view. The signal with positive Hela l-CaD staining extends well beyond the cell surface as defined by the expression of CD34 and CD133. This area is designated as lamellipodium, containing filopodia and microspikes. (B) The cell is consecutively scanned by optical sectioning at z-stacks (3D) for confirming the validity of the observation at different levels in the cell. The signals of Hela l-CaD-positive bundles as microspikes or filopodia are invariably present. Scale bars = 20 µm.

Figure 5.

Hela l-CaD and fragmented F-actin in the cores of podosomes. (A) Scanning is performed at the single cell level. Hela l-CaD is present in a punctuated pattern in the leading lamella of the cell (CD34 channel not shown). (B) A region in (A) is highlighted. Hela l-CaD is seen in dot-like structures and colocalizes with talin. Talin expression extends well beyond (and surrounds) the dots. (C) Cell scanned as (A). F-actin is fragmented at the cell periphery representing “belt podosomes”. (D) A region in (C) is highlighted. The fragmented F-actin as “cores” are surrounded by talin. (E) Cell scanned as (A and B). F-actin is fragmented in the leading lamella of this cell (CD34 channel not shown). (F) Highlighted region of E. Vinculin predominantly surrounds the “actin cores”. (G) Cell scanned as (A, C and E). F-actin is fragmented in the leading lamella of this cell (KDR channel not shown). (H) Gelsolin colocalizes with the “actin cores”. (I) TM predominantly appears in the surrounding of the actin cores. Scale bars = 50µm.

Figure 6.

Differential expression of gelsolin in normal vascular components. (A) Gelsolin is expressed in smooth muscle cells, not ECs, in the wall of a blood vessel in normal brain (x 400). (B) Double labeling for CD34 and gelsolin reveals separate localizations of these proteins, indicating that gelsolin is not expressed in the lining of endothelial cells. (C) Overlapping expression of SMA and gelsolin, which confirms the finding in (B). Scale bars (B and C) = 20 µm.

Figure 7.

Hela l-CaD accumulated in membrane blebs of an EPC in a glioblastoma. The surface of the cell is delineated by immunopositivity for CD34 and KDR. Hela l-CaD is present beyond the cell surface in lamellipodium accumulating as small oval cell protrusions. Scale bar = 50 µm.

Figure 8.

Hela l-CaD in a membrane ruffle of an EC in a breast cancer. An irregular and regional protrusion of the cell membrane characteristic of a membrane ruffle is observed. KDR staining is negative (not shown). Scale bars = 20 µm.

Discussion

In this study the features and protein expression patterns of motile cells as described in in vitro studies^3,29,38–41 were detected in the EPCs/ECs of the tumor tissues in situ. The Hela l-CaD-containing cell protrusions are specific for tumor ECs/EPCs and have never been observed in normal ECs. Quantitative heterogeneity of this phenomenon is, to some extent, observed between different tumors. The occurrence of a variety of asymmetric cell protrusions in which reorganization of the CSK plays a pivotal role is characteristic of cell migration.⁴² However, the characteristics of cells in vitro may not be representative of those encountered in vivo. It is evident that cell culture (in vitro) experiments would perturb ECs from their quiescent in vivo state (0.1% replications per day) to an activated phenotype (1% to 10% replications per day) with definite loss of specialized cell functions.⁴³ There is no model for generating resting ECs in vitro.⁴³ So far, only in vitro data on the role of caldesmon in motile cells exist in the literature.^44–45 The present study was specifically designed to identify the Hela l-CaD isoform in motility-related structures of human ECs/EPCs on the tumor tissues in situ (histologically preserved microenviroment close to a life-like state). To the best of our knowledge, this is the first study in which the morphological features of cell motility were captured in situ in human tumor specimens.

Hela l-CaD is specifically expressed in the subregions of all types of cell protrusions and podosomes, suggesting that this molecule is implicated in the migration of the ECs/EPCs in vivo. In cultured and transfected cells the overexpression of the actin-binding domain, or full length, of l-CaD promotes cell movement and facilitates the formation of cytoplasmic processes, while cell contractility is inhibited and the number of focal adhesions are decreased.⁴⁶ These l-CaD-associated phenomena may well result from the reorganization of actomyosin-associated molecules to form actin-rich dynamic cell protrusions. Indeed, in in vitro studies, l-CaD has been observed in membrane ruffles,³⁸ podosomes,³ lamellipodia³⁹ and membrane blebs⁴⁰ which are typically associated with cell migration.^31,47,48 Our findings support the notion that the features of motile cells which were described in vitro have their counterparts in the activated ECs/EPCs in tissue samples of human tumors. The bundling of CaD as microspikes or filopodia has not yet been reported in in vitro studies so far. Interestingly, Hela l-CaD-positive multinucleated ECs/EPCs were relatively often seen in the tumor samples, particularly in the glioma samples. Multinucleation is believed to be brought about by endomitosis and considered as a sign of aborted cytokinesis⁴⁹ with concomitant activation of motility and podosome formation.⁵⁰ The activated motility of these multinuclear cells may well explain their ubiquitous distribution in the tumor tissue sections. A variety of studies have shown that phosphorylation of caldesmon plays an important role in cytoskeleton remodeling during cell cycle and migration.^51,52 The overlap of expression patterns of Hela l-CaD and pCaD supports the notion that Hela l-CaD detected in these protrusive regions is a functional protein.

In Hela l-CaD-positive ECs/EPCs the disassembly of vinculin and talin-containing focal adhesions (FAs) always coincides with the reorganization of F-actin and the formation of podosomes. This phenomenon is similar to that observed in in vitro studies.⁵⁰ In in vivo studies on motile cells it has been shown that vinculin and talin are required for strong adhesion and cytoskeletal organization and also serve functions in cell migration.^53,54 This observation was corroborated by the deficits induced in talin- and vinculin knockouts, which consisted of failure of cell migration and cell adhesion.^53,54 Podosomes are highly dynamic adhesion structures consisting of a variety of protein components, which are present as a clustered complex. A podosome is detected by the presence of a cluster of particular proteins, not by the presence of any of these single proteins alone.⁵⁵ In the podosomes, F-actin and l-CaD are seen as dot-like structures or form dense marginal rings at the leading edges in the cell periphery. In normal vessels, vinculin/talin-based FAs are located at the basal membrane region of the endothelium, perpendicularly to the abluminal site.⁵ We found that vinculin and talin redistribute into the cytoplasm and join the formation of podosomes as described in cultured cells.³¹ These findings confirm the dramatic structural changes of the EC/EPC cytoskeleton during the transition from the quiescent to the activated state. Similar to what has been described in in vitro studies,³ we did not find TM in the core domain of podosomes. Actin serving proteins like gelsolin play an important role in regulating the number of filaments and the filament length by tightly binding to the barbed ends in order to avoid elongation.⁵⁶ Gelsolin is one of the best characterized actin serving proteins⁵⁷ and is required for rapid movements and signal transduction in cells responding to stress.¹⁸ In resting cells, gelsolin is either inactive or associated as capper of filaments.⁵⁸ In this study, we were unable to find gelsolin in normal vascular ECs, but found it in the leading lamellae and podosomes of Hela l-CaD-positive ECs/EPCs, indicative of activation of migration of these cells. The detection of cortactin and Arp2/3 in the core domains of podosomes is consistent with in vitro results.³⁰

In conclusion, the abnormal expression of the Hela l-CaD with its specific subcellular distribution coincides with the appearance of cellular features of locomotion in ECs/EPCs of tumor vasculature. The data not only provide novel insights into the regulation of the actin-associated proteins in tumor neovascularization, but may also direct the development of novel therapeutic agents targeted toward arrest of pathologic neovascularization.