**2. Method of culture of VSMCs in type I collagen three-dimensional "honeycomb" matrices**

We reported a method for culturing contractile VSMCs by using three-dimensional matrices, so-called "honeycombs." Honeycombs are type I collagen sponges that can be obtained from Koken Co., Ltd. (Japan). Honeycombs are prepared from 0.5% type I atelocollagen in an acid solution by neutralization with ammonia gas to separate the collagen fibrils and lyophilization [3]. The structure of the honeycomb is porous and consists of many tubes aligned side by side, similar to a beehive [4, 5]. The pore diameter of the honeycomb is controlled by altering the concentration of the collagen solution and ammonia gas. When a higher concentration of ammonia gas is used, smaller pores are produced, and vice versa [3]. The diameter of each pore of the tubes is 100–500 µm, and we usually use a pore size of 200–300 µm for rabbit and human VSMCs. The honeycombs are cut vertically into cubes of dimensions 5 × 5 × 2 or 3 × 3 × 2 mm for culturing VSMCs.

The initial culture of VSMCs in a honeycomb is as follows (Figure 1): rabbit or mouse VSMCs cultured on plates (synthetic phenotype) in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS are used. The honeycombs are pre-incubated with DMEM containing 10% FBS and the air in each pore of the honeycomb is removed by centrifugation. VSMCs cultured on plates are incubated with trypsin-EDTA, and the released cells are collected by centrifuga‐ tion. The collected cells are suspended in 300–400 µL DMEM containing 10% FBS and are incubated with the honeycombs (approximately 2.0 – 3.0 × 106 cells per 30 honeycombs) on a dish (diameter, 6 cm) for 3 h at 37°C. Then, 3–5 mL culture medium is added. The medium is changed every 2–3 days. For human VSMCs, a commercial medium supplemented with epidermal growth factor, fibroblast growth factor-B, insulin, and 2% FBS is used.

**1. Introduction**

442 Muscle Cell and Tissue

depends on the surrounding environment [1].

system for VSMCs that maintain their contractile phenotype.

**"honeycomb" matrices**

× 2 mm for culturing VSMCs.

Vascular smooth muscle cells (VSMCs) are the major cell type in the vascular wall and their main role is contraction. VSMCs in the normal aorta are classified as having a contractile phenotype because they contract without proliferation. Conversely, VSMCs in vascular diseases, such as restenosis after percutaneous coronary intervention and the formation of atherosclerotic plaques, are described as having a synthetic phenotype because they can proliferate and migrate, but lose their ability to contract. The phenotypic modulation of VSMCs

In order to conduct basic studies on the physiological function of VSMCs and to develop novel medical treatments, cultured VSMCs are used. When VSMCs are cultured on a plastic plate in the presence of 10% fetal bovine serum (FBS), which is the normal two-dimensional monolayer culture system, the cells can migrate and proliferate, but lose their contractile ability [2]. Cultured VSMCs are classified as having a synthetic phenotype and are used as a model of atherosclerotic lesion cells. As phenotypic modulation of VSMCs is responsible for restenosis and the progression of atherosclerosis, they could be the main target of medicinal treatment, and VSMCs cultured on plates are useful to evaluate the effects of medicines. However, there is no acceptable cell model that reflects the nature of VSMCs in the normal aorta of a living body. For this reason, information generated from synthetic VSMCs cannot be compared with that from contractile VSMCs in the normal aorta. In this chapter, we describe a new culture

**2. Method of culture of VSMCs in type I collagen three-dimensional**

We reported a method for culturing contractile VSMCs by using three-dimensional matrices, so-called "honeycombs." Honeycombs are type I collagen sponges that can be obtained from Koken Co., Ltd. (Japan). Honeycombs are prepared from 0.5% type I atelocollagen in an acid solution by neutralization with ammonia gas to separate the collagen fibrils and lyophilization [3]. The structure of the honeycomb is porous and consists of many tubes aligned side by side, similar to a beehive [4, 5]. The pore diameter of the honeycomb is controlled by altering the concentration of the collagen solution and ammonia gas. When a higher concentration of ammonia gas is used, smaller pores are produced, and vice versa [3]. The diameter of each pore of the tubes is 100–500 µm, and we usually use a pore size of 200–300 µm for rabbit and human VSMCs. The honeycombs are cut vertically into cubes of dimensions 5 × 5 × 2 or 3 × 3

The initial culture of VSMCs in a honeycomb is as follows (Figure 1): rabbit or mouse VSMCs cultured on plates (synthetic phenotype) in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS are used. The honeycombs are pre-incubated with DMEM containing 10% FBS and the air in each pore of the honeycomb is removed by centrifugation. VSMCs cultured on plates are incubated with trypsin-EDTA, and the released cells are collected by centrifuga‐

**Figure 1.** Preparation of VSMCs into honeycombs. Rabbit, mouse, and human VSMCs can be cultured successfully in honeycombs with this chart. The required number of cells are cultured on a plate as synthetic VSMCs and transferred to the honeycombs. To collect VSMCs in honeycombs for analysis, the honeycombs are treated with collagenase-I.

By visualizing the binding process of VSMCs to honeycombs using a real-time cultured cell monitoring system [6], VSMCs are fixed at one attachment point and move little by little using other attachment points, resembling the movement of an inchworm. After 25 h incubation, the positions of the VSMCs are fixed, and they form cross-bridges in the honeycombs. VSMCs in honeycombs form 2–5 attachment points per cell (Figure 2). However, mouse VSMCs (length of long axis, 33–139 [80 ± 29] µm) do not make cross-bridges in honeycombs in which the diameter of the pore is about 500 µm. These results suggest that the relationship between pore size and the length of the long axis of VSMCs is important for the formation of cellular crossbridges. The inner wall of honeycombs with a larger pore size may function as a "flat floor" for VSMCs, as if it were a plastic plate.

size may function as a "flat floor" for VSMCs, as if it were a plastic plate.

Figure 2. Cross-bridges of VSMCs on honeycombs. These pictures are electron microscopic observations of rabbit and mouse VSMCs cultured in honeycombs. (A) Rabbit VSMCs cultured in honeycombs of pore size ≤200 µm; (B) rabbit VSMCs cultured in honeycombs of pore size 300–500 µm; (C) mouse VSMCs **Figure 2.** Cross-bridges of VSMCs on honeycombs. These pictures are electron microscopic observations of rabbit and mouse VSMCs cultured in honeycombs. (A) Rabbit VSMCs cultured in honeycombs of pore size ≤200 µm; (B) rabbit VSMCs cultured in honeycombs of pore size 300–500 µm; (C) mouse VSMCs cultured in honeycombs of pore size ≤200 µm; and (D) mouse VSMCs cultured in honeycombs of pore size 300–500 µm. Arrows show VSMCs. Scale bars, 50 µm. Data adapted from reference 6.

cultured in honeycombs of pore size ≤200 µm; and (D) mouse VSMCs cultured

#### in honeycombs of pore size 300–500 µm. Arrows show VSMCs. Scale bars, 50 **3. Proliferative inhibition of VSMCs cultured in honeycombs**

µm. Data adapted from reference 6. 3. Proliferative inhibition of VSMCs cultured in honeycombs Various kinds of cells, such as human fibroblasts, CHO-K1, BHK-21, and bovine endothelial cells [3], can be cultured successfully in honeycombs. These cells grow normally in honey‐ combs. However, the proliferation of VSMCs can be controlled easily by the pore size of the honeycombs.

Various kinds of cells, such as human fibroblasts, CHO-K1, BHK-21, and bovine endothelial cells (3), can be cultured successfully in honeycombs. These cells grow normally in honeycombs. However, the proliferation of VSMCs can be controlled easily by the pore size of the honeycombs. When synthetic rabbit VSMCs are used for culture in honeycombs (pore size, about 200 µm), they stop proliferating immediately when they form cross-bridges (4). [<sup>3</sup>H]thymidine is incorporated at a low level into VSMCs cultured in honeycombs, and cell number does not change during When synthetic rabbit VSMCs are used for culture in honeycombs (pore size, about 200 µm), they stop proliferating immediately when they form cross-bridges [4]. [3 H]Thymidine is incorporated at a low level into VSMCs cultured in honeycombs, and cell number does not change during culture. VSMCs can be cultured in honeycombs for approximately 3 months with medium change. However, when rabbit VSMCs are cultured in honeycombs with larger pore size (100–500 µm) (Figure 3), they proliferate for the first few days, but then stop proliferating and cell number does not change [6]. The reason why cell number increases only at the beginning of culture is as follows: in honeycombs with a smaller pore (≤200 µm), VSMCs form cross-bridges independently of each other, but a large number of connected cells form cross-bridges together at the wall of honeycombs with larger pores (100–500 µm) after the initial increase in cell number at the beginning of culture. These data suggest that the formation

5

of cross-bridges by VSMCs in honeycombs may be a significant step for the cessation of proliferation.

Figure 3. Cross-bridges of rabbit VSMCs cultured in honeycombs for 14 days. Electron microscopic observation of rabbit VSMCs cultured in honeycombs for 14 days. Rabbit VSMCs were cultured in honeycombs of pore size 100–500 µm. **Figure 3.** Cross-bridges of rabbit VSMCs cultured in honeycombs for 14 days. Electron microscopic observation of rab‐ bit VSMCs cultured in honeycombs for 14 days. Rabbit VSMCs were cultured in honeycombs of pore size 100–500 µm. The pores enclosed by squares contain a large number of VSMCs, and the pores enclosed by circles contain a small number of VSMCs forming cross-bridges. Arrows show VSMCs. Scale bar, 100 µm. Data adapted from reference 6.

The pores enclosed by squares contain a large number of VSMCs, and the pores enclosed by circles contain a small number of VSMCs forming cross-bridges. Arrows show VSMCs. Scale bar, 100 µm. Data adapted from reference 6. When VSMCs are cultured on collagen-coated plates, their proliferation rate increases. Conversely, when VSMCs are cultured on collagen gels, their proliferation stops via the cdk2 inhibitor p27kip1 [7]. However, proliferative inhibition persists for less than 1 week because VSMCs dissolve the collagen gel and start to proliferate again (unpublished data). Therefore, it is expected that both the higher-order structure of collagen and pore size of the honeycombs are considerable factors for the proliferative inhibition of VSMCs.

When VSMCs are cultured on collagen-coated plates, their proliferation rate increases. Conversely, when VSMCs are cultured on collagen gels, their proliferation stops via the cdk2 inhibitor p27kip1 (7). However, proliferative inhibition persists for less than 1 week because VSMCs dissolve the collagen gel and start to proliferate again (unpublished data). Therefore, it is expected that both the higher-order structure of collagen and pore size of the honeycombs are considerable factors for the Although VSMCs in honeycombs stop proliferating immediately, p27kip1 expression increases after incubation for 2–3 days (unpublished data). From this observation, it is expected that p27kip1 may work to keep VSMCs at a resting state and not as the initiator of proliferative inhibition. Taken together with electron microscopic observations and data for growth in honeycombs, proliferative inhibition occurs in parallel with a decrease in the number of focal adhesions, including reduced levels of focal adhesion kinase (FAK). As a high level of phosphorylated FAK promotes proliferative activity [8], the low level of phosphorylated FAK is one of the reasons for proliferative inhibition of VSMCs in honeycombs.

7

proliferative inhibition of VSMCs.

5

of cellular cross-bridges. The inner wall of honeycombs with a larger pore

Figure 2. Cross-bridges of VSMCs on honeycombs. These pictures are electron microscopic observations of rabbit and mouse VSMCs cultured in honeycombs. (A) Rabbit VSMCs cultured in honeycombs of pore size ≤200 µm; (B) rabbit VSMCs cultured in honeycombs of pore size 300–500 µm; (C) mouse VSMCs cultured in honeycombs of pore size ≤200 µm; and (D) mouse VSMCs cultured in honeycombs of pore size 300–500 µm. Arrows show VSMCs. Scale bars, 50

**Figure 2.** Cross-bridges of VSMCs on honeycombs. These pictures are electron microscopic observations of rabbit and mouse VSMCs cultured in honeycombs. (A) Rabbit VSMCs cultured in honeycombs of pore size ≤200 µm; (B) rabbit VSMCs cultured in honeycombs of pore size 300–500 µm; (C) mouse VSMCs cultured in honeycombs of pore size ≤200 µm; and (D) mouse VSMCs cultured in honeycombs of pore size 300–500 µm. Arrows show VSMCs. Scale bars, 50 µm.

Various kinds of cells, such as human fibroblasts, CHO-K1, BHK-21,

H]Thymidine is

When synthetic rabbit VSMCs are used for culture in honeycombs

and bovine endothelial cells (3), can be cultured successfully in honeycombs. These cells grow normally in honeycombs. However, the proliferation of

(pore size, about 200 µm), they stop proliferating immediately when they form cross-bridges (4). [<sup>3</sup>H]thymidine is incorporated at a low level into VSMCs cultured in honeycombs, and cell number does not change during

VSMCs can be controlled easily by the pore size of the honeycombs.

3. Proliferative inhibition of VSMCs cultured in honeycombs

Various kinds of cells, such as human fibroblasts, CHO-K1, BHK-21, and bovine endothelial cells [3], can be cultured successfully in honeycombs. These cells grow normally in honey‐ combs. However, the proliferation of VSMCs can be controlled easily by the pore size of the

When synthetic rabbit VSMCs are used for culture in honeycombs (pore size, about 200 µm),

incorporated at a low level into VSMCs cultured in honeycombs, and cell number does not change during culture. VSMCs can be cultured in honeycombs for approximately 3 months with medium change. However, when rabbit VSMCs are cultured in honeycombs with larger pore size (100–500 µm) (Figure 3), they proliferate for the first few days, but then stop proliferating and cell number does not change [6]. The reason why cell number increases only at the beginning of culture is as follows: in honeycombs with a smaller pore (≤200 µm), VSMCs form cross-bridges independently of each other, but a large number of connected cells form cross-bridges together at the wall of honeycombs with larger pores (100–500 µm) after the initial increase in cell number at the beginning of culture. These data suggest that the formation

**3. Proliferative inhibition of VSMCs cultured in honeycombs**

they stop proliferating immediately when they form cross-bridges [4]. [3

µm. Data adapted from reference 6.

Data adapted from reference 6.

honeycombs.

444 Muscle Cell and Tissue

size may function as a "flat floor" for VSMCs, as if it were a plastic plate.

An additional mechanism for the proliferative inhibition of VSMCs in this three-dimensional culture system is the expression of ornithine decarboxylase antizyme 1 (OAZ1), which is a key regulator of intracellular polyamines. Polyamines (putrescine, spermidine, and spermine), which are multivalent organic cations, are essential for cell growth [9]. The proliferation and transformation of cells induced by oncogenes, carcinogens, and viruses are characterized by increases in the levels of intracellular polyamines due to their increased biosynthesis and uptake [10]. Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosyn‐ thesis. OAZ1 inhibits the activity of ODC and increases its degradation by forming an OAZ1- ODC complex [11, 12]. OAZ1 also decreases the uptake of polyamines independent of its effects on ODC [13, 14].

When VSMCs proliferate on plates, the intracellular content of polyamines increases. Con‐ versely, polyamine content is maintained at a low level in VSMCs cultured in honeycombs [15]. VSMCs stably transfected with the ODC gene (ODC-VSMCs) and cultured on plates increase their rate of proliferation, which is accompanied by an increase of polyamine content (espe‐ cially spermidine) and phosphorylated FAK and a decrease in marker proteins of differentia‐ tion, α-actin, and myosin heavy chain in comparison to VSMCs cultured on plates (Figure 4). As ODC is an oncogene and induces an excess of polyamines [16], it is assumed that ODC may promote the proliferative activity of VSMCs cultured in honeycombs. However, the prolifer‐ ation of ODC-VSMCs also ceases in honeycombs, similar to normal VSMCs with low levels of spermidine and phosphorylated FAK (Figure 4). Finally, culture of ODC-VSMCs in honey‐ combs increases the expression of α-actin and myosin heavy chain.

As shown in Figure 4, OAZ1 levels are higher in both VSMCs and ODC-VSMCs cultured in honeycombs than in both VSMCs and ODC-VSMCs cultured on plates. OAZ1 is degraded in ODC-VSMCs cultured on plates for up to 3 h after treatment with cycloheximide, but OAZ1 degradation in ODC-VSMCs cultured in honeycombs is limited over a 12 h incubation period (Figure 4). This difference in OAZ1 stability may be the major reason for the high levels of OAZ1 observed in VSMCs cultured in honeycombs. These results suggest that OAZ1 in VSMCs cultured in honeycombs might enhance the degradation of ODC and inhibit polyamine uptake. In fact, our data show that polyamine uptake is significantly lower in ODC-VSMCs cultured in honeycombs than in ODC-VSMCs cultured on plates [15]. This could be one of the reasons for the lower polyamine levels in VSMCs cultured in honeycombs than in VSMCs cultured on plates.

When OAZ1 is transiently overexpressed in ODC-VSMCs (Figure 5), OAZ1 decreases the number of ODC-VSMCs cultured on plates at day 3, whereas OAZ1 overexpression does not change the number of ODC-VSMCs cultured in honeycombs. OAZ1 overexpression slightly decreases the expression of ODC and phosphorylated FAK levels in ODC-VSMCs cultured on plates. Surprisingly, OAZ1 overexpression influences the levels of myosin heavy chain in ODC-VSMCs cultured on plates and in honeycombs, and influences the levels of α-actin in ODC-VSMCs cultured in honeycombs. These results suggest that OAZ1 in VSMCs might regulate intracellular polyamine levels and cellular proliferation. Moreover, OAZ1 levels may influence the expression of α-actin and myosin heavy chain, which are the main components of the contractile apparatus.

Three-Dimensional "Honeycomb" Culture System that Helps to Maintain the Contractile Phenotype of… http://dx.doi.org/10.5772/60960 447

An additional mechanism for the proliferative inhibition of VSMCs in this three-dimensional culture system is the expression of ornithine decarboxylase antizyme 1 (OAZ1), which is a key regulator of intracellular polyamines. Polyamines (putrescine, spermidine, and spermine), which are multivalent organic cations, are essential for cell growth [9]. The proliferation and transformation of cells induced by oncogenes, carcinogens, and viruses are characterized by increases in the levels of intracellular polyamines due to their increased biosynthesis and uptake [10]. Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosyn‐ thesis. OAZ1 inhibits the activity of ODC and increases its degradation by forming an OAZ1- ODC complex [11, 12]. OAZ1 also decreases the uptake of polyamines independent of its effects

When VSMCs proliferate on plates, the intracellular content of polyamines increases. Con‐ versely, polyamine content is maintained at a low level in VSMCs cultured in honeycombs [15]. VSMCs stably transfected with the ODC gene (ODC-VSMCs) and cultured on plates increase their rate of proliferation, which is accompanied by an increase of polyamine content (espe‐ cially spermidine) and phosphorylated FAK and a decrease in marker proteins of differentia‐ tion, α-actin, and myosin heavy chain in comparison to VSMCs cultured on plates (Figure 4). As ODC is an oncogene and induces an excess of polyamines [16], it is assumed that ODC may promote the proliferative activity of VSMCs cultured in honeycombs. However, the prolifer‐ ation of ODC-VSMCs also ceases in honeycombs, similar to normal VSMCs with low levels of spermidine and phosphorylated FAK (Figure 4). Finally, culture of ODC-VSMCs in honey‐

As shown in Figure 4, OAZ1 levels are higher in both VSMCs and ODC-VSMCs cultured in honeycombs than in both VSMCs and ODC-VSMCs cultured on plates. OAZ1 is degraded in ODC-VSMCs cultured on plates for up to 3 h after treatment with cycloheximide, but OAZ1 degradation in ODC-VSMCs cultured in honeycombs is limited over a 12 h incubation period (Figure 4). This difference in OAZ1 stability may be the major reason for the high levels of OAZ1 observed in VSMCs cultured in honeycombs. These results suggest that OAZ1 in VSMCs cultured in honeycombs might enhance the degradation of ODC and inhibit polyamine uptake. In fact, our data show that polyamine uptake is significantly lower in ODC-VSMCs cultured in honeycombs than in ODC-VSMCs cultured on plates [15]. This could be one of the reasons for the lower polyamine levels in VSMCs cultured in honeycombs than in VSMCs

When OAZ1 is transiently overexpressed in ODC-VSMCs (Figure 5), OAZ1 decreases the number of ODC-VSMCs cultured on plates at day 3, whereas OAZ1 overexpression does not change the number of ODC-VSMCs cultured in honeycombs. OAZ1 overexpression slightly decreases the expression of ODC and phosphorylated FAK levels in ODC-VSMCs cultured on plates. Surprisingly, OAZ1 overexpression influences the levels of myosin heavy chain in ODC-VSMCs cultured on plates and in honeycombs, and influences the levels of α-actin in ODC-VSMCs cultured in honeycombs. These results suggest that OAZ1 in VSMCs might regulate intracellular polyamine levels and cellular proliferation. Moreover, OAZ1 levels may influence the expression of α-actin and myosin heavy chain, which are the main components

combs increases the expression of α-actin and myosin heavy chain.

on ODC [13, 14].

446 Muscle Cell and Tissue

cultured on plates.

of the contractile apparatus.

Figure 4. OAZ1 in normal VSMCs and ODC-VSMCs, and the levels of various proteins expressed in VSMCs cultured on plates and in honeycombs. (A) (a) **Figure 4.** OAZ1 in normal VSMCs and ODC-VSMCs, and the levels of various proteins expressed in VSMCs cultured on plates and in honeycombs. (A) (a) VSMCs were transfected with three kinds of pTracer-CMV containing OAZ1 without T205, which do not need a frameshift for their expression of each type of OAZ1. Lane 1, pTracer-OAZ∆T205∆AUG1, which expresses 24.5-kDa OAZ1; lane 2, pTracer-OAZ∆T205, which expresses 24.5- and 29-kDa

VSMCs were transfected with three kinds of pTracer-CMV containing OAZ1

without T205, which do not need a frameshift for their expression of each type of OAZ1. Lane 1, pTracer-OAZ∆T205∆AUG1, which expresses 24.5-kDa OAZ1;

lane 2, pTracer-OAZ∆T205, which expresses 24.5- and 29-kDa OAZ1; lane 3,

pTracer-OAZ∆T205∆AUG2, which expresses 29-kDa OAZ1. After transfection,

11

OAZ1; lane 3, pTracer-OAZ∆T205∆AUG2, which expresses 29-kDa OAZ1. After transfection, VSMCs were collected at 24 h, a cell extract was prepared, and Western blot analysis was performed. (b) Degree of inhibition of ODC by OAZ1 was measured. The activity of OAZ1 was determined as an inhibitory percentage of ODC activity of the extract from ODC-overproducing FM3A (EXOD-1) cells. ●, extract from VSMCs expressing 24.5-kDa OAZ1; ▲, extract from VSMCs expressing 29- and 24.5-kDa OAZ1; and ○, extract from VSMCs expressing 29-kDa OAZ1. Values are means of duplicate determinations. (B) Inhibition of ODC activity using extracts from VSMCs cultured on plates and in honey‐ combs. OAZ1 activity was determined as an inhibitory percentage of ODC activity of the extract from EXOD-1 cells. Values are means ± standard deviation (SD) of triplicate determinations. (C) Western blot analysis of OAZ1, ODC, ty‐ rosine-phosphorylated FAK (pY-FAK), α-actin, myosin heavy chain, and β-actin of VSMCs and ODC-VSMCs cultured on plates and in honeycombs. P, EXOD-1 cells as a positive control. Relative intensity on day 7 was quantified. The intensity of OAZ1 was quantified as the sum of the 29- and 24.5-kDa bands. ND, not detectable. Values are means ± SD of triplicate determinations. (D) OAZ1 degradation. ODC-VSMCs cultured on plates and in honeycombs for 3 days were treated with 20 µg/mL cycloheximide for the indicated time. Data adapted from reference 15.

VSMC proliferation is reportedly up-regulated via an increase in the stability of S-phase kinaseassociated protein-2, E3 ubiquitin protein ligase (SKP2) by autophosphorylation of FAK-Tyr397 [17]. In ODC-VSMCs cultured on plates, the increased rate of proliferation is accompanied by an increase of phosphorylated FAK. Interestingly, when the effect of spermine on FAK autophosphorylation *in vitro* was investigated, FAK phosphorylation was stimulated by spermine [15]. The low levels of intracellular polyamines in VSMCs cultured in honeycombs could inhibit the autophosphorylation of FAK, and this may contribute to the proliferative inhibition of VSMCs.
