Assay Techniques 41 Identification of VSMCs

Because of the heterogeneity and adaptive modulation displayed by VSMCs in culture, a number of criteria must be used to ascertain that cultured cells are derived from VSMCs. This is particularly important for cells derived from atherosclerotic vessels, as some cell types such as myofibroblasts and primitive mesenchymal cells share expression of a number of SM-contractile proteins with VSMCs. Indeed, there are no definitive markers to differentiate between these cell types and VSMCs. Therefore, in order to determine that a particular culture is VSMC-derived, the researcher must use a number of criteria including origin of the cells, morphology and expression of a 'battery' of smooth muscle markers. To date, many of these markers have been derived from studies in other animal species, and there is need for further identification of more appropriate and specific markers of human VSMC phenotypes in vitro.

4.1.1 Morphology

Cultured human VSMCs are morphologically heterogeneous. Typically, as VSMCs approach confluence, they exhibit a 'hills and valleys' morphology where cells make contacts with each other and retract. However, VSMCs can also appear as elongate, spindle, cobblestone or large, rounded senescent cells. Examples of these various morphologies are shown in Figure 2. As mentioned previously, these types of VSMCs are not necessarily stable in culture, and we have found that the small, primary cells shown in Figure 2F can change their morphological appearance after passaging. Occasionally, it has also been noted that some cells migrating from explants contain lipid droplets which appear as small cytoplasmic vesicles (using phase-contrast microscopy) and stain positively with Oil-Red O. In addition, electron microscopy can be used to analyze features of VSMCs; cells with a more 'contractile' phenotype appearing as a cell with a heterochromatic nucleus and abundant actin and myosin filaments, compared with 'synthetic' VSMCs with a euchromatic nucleus, prominent sarcoplasmic reticulum and Golgi complex (34).

4.1.2 Multicellular nodule formation by VSMCs

The capacity for human VSMCs to form 3D nodules or spheroids at postconfluence (as shown in Figure 2) has been well described for arterial VSMCs [17, 21, 35, 36] and pericytes [37]. We have found that human arterial VSMCs (aortic, tibial and femoral) and human placental pericytes form nodules in culture which calcify after approximately 30 days [21, 38]. This property of VSMCs exists in almost all isolates that we have prepared. The only morphological-type which does not form nodules are the 'cobblestone' cells which appear to be contact-inhibited. Although not unique to VSMCs (mesenchymal cells with 'osteoblastic properties' may also form nodules), this property will distinguish VSMCs from fibroblasts.

4.1.3 VSMC contraction

A unique property of VSMCs compared with other cell types present in blood vessels is contractility. However, measuring contraction in cultured cells is not straightforward, as VSMCs lose many of their contractile properties with time in culture. Therefore, most studies of VSMC contraction involve culture of blood vessel ring segments that retain some contractile responses, and these can be measured by treatment with agonists such as noradrenaline or KCI and subsequent measurement of contractile force and/or intracellular free calcium concentration. However, in a study by Shirinsky et al., contraction of enzyme-dispersed rabbit VSMCs occurred in response to histamine [39]. Contraction was observed during the first six days of culture and was measured optically. Measurement of aspects of VSMC contraction in culture is therefore feasible. More recently, contraction of individual human VSMCs in culture has been reported [39a].

4.2 Cell markers 4.2.1 Antibodies a-smooth muscle (aSM) actin has been universally used as a marker for VSMCs [40]. However, some studies have reported that endothelial cells and adventitial fibroblasts can express a-SM actin under certain culture conditions [41, 42]. Therefore, a battery of SM markers must be used to identify cells in vitro. Most VSMC markers are contractile proteins, and antibodies for many of these are widely available commercially. Some common markers include; smooth muscle myosin heavy chain (SMMHC), calponin, SM22a, desmin, h-caldesmon, metavinculin and smoothelin. SMMHC is the most specific marker. However, expression of this protein is not always maintained in SM cultures. In general, to confirm that cultured cells derived from the vessel media are VSMCs, we require positivity for aSM actin and a least one other SM contractile protein (e.g. calponin or SM22a). Cells are tested by immunohistochemistry, which requires very few cells grown in multi-well chamber slides, fixed in paraformaldehyde. Alternatively, Western analysis can be performed on cell lysates.

It may also be appropriate to confirm that cultures are von Willebrand factor negative if there is concern about endothelial cell contamination (for example, when isolating VSMCs from small blood vessels). Separate markers have also been described for microvascular SMCs (pericytes) such as 3G5 [43], but there is debate about the specificity of this marker [44]. The high molecular weight melanoma-associated antigen is also a marker for pericytes in vivo [28].

4.2.2 Molecular Profile

Gene expression studies are probably the most reliable method for determining the type of cell that has been cultured (although they generally cannot detect contamination of a culture with another cell type). mRNA can be extracted from cultures and gene expression can be analysed by either Northern or RT-PCR. The most reliable markers for analysis are again the SM-specific contractile proteins including a-SM actin, SM22a and calponin, which are easily detectable on Northern analysis. SMMHC may be detectable by Northern analysis, but RT-PCR may be necessary. To date, for human cells, there are no markers that will definitively identify a VSMC. However, expression of a number of SM contractile proteins detectable by Northern analysis is sufficient, particularly if the anatomical source of the material is certain [45].

4.2.3 Extracellular matrix

As described in the introduction, VSMCs in vivo are surrounded by various extracellular matrix components. Extensive studies of the types of matrix secreted by human VSMCs in vitro have not yet been performed. In other species, VSMCs secrete various collagen types, fibronectin, laminin, proteoglycans, elastin, and SPARC, as well as integrin receptors for binding to extracellular matrix (reviewed in 34). Human VSMCs synthesize matrix degrading enzymes as well as endogenous inhibitors of matrix degradation [46, 47]. Although these extracellular matrix proteins are not unique to VSMCs, the expression in culture of a large subset of these matrix components, combined with expression of a SM contractile marker, may be indicative that the cells are SM in origin.

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