Functional effects of B. henselae infection on MAC pro-angiogenic activity

5.2.1Spheroid assay of sprouting angiogenesis

As an intermediary between monolayer cell culture and in vivo assays, the endothelial spheroid assay of sprouting angiogenesis provides a reproducible and easily manipulated culture environment that more closely mimics the complexity of tissue in vivo (Pampaloni et al., 2007). In the absence of suitable animal models, the spheroid assay is a reasonable setting to examine the effects of B. henselae infection on multiple molecular events and cellular interactions that make up in vivo angiogenic process.

In the spheroid assay cells form spherical aggregates and under angiogenic conditions develop radial sprouts which mimic the process of tip cell activation, tip-versus-stalk cell selection, matrix degradation, and chemotactic sprout extension. (Korff and Augustin, 1999). Importantly, the 3D spheroid culture environment provides realistic cell-cell and cell-matrix interactions which have strong effects on cellular phenotype and functional capacity (Fennema et al., 2013).

The spheroid assay has previously been used to model B. henselae induced pathological angiogenesis in endothelial cells. Scheidegger et. al. (2009) demonstrated that infection of HUVECs with B. henselae prior to spheroid formation increased the rate of angiogenic sprouting significantly.

Activities of the VirB/VirD T4SS translocated effector proteins were found to modulate the rate of infection associated angiogenic sprouting. BepA, which is strongly associated with B. henselae induced inhibition of apoptosis, was shown to increase angiogenic sprouting whereas BepG, which is associated with cytoskeletal rearrangement and invasome formation, was shown to decrease the rate of vascular sprouting.

To investigate how B. henselae infection may influence the interaction of MACs with the vascular endothelium, a co-culture spheroid model of HUVECs and MACs was developed for the first time.

The pro-angiogenic activities of MAC in vivo are dependent on their ability to home to sites of angiogenic activity and interact with the vascular endothelium. MACs are recruited to the site of neo-vascularization or repair in a multistep process involving maturation and release form the bone marrow, chemoattraction via cytokines (such as SDF-1 and CCL2), adhesion to the vascular wall (via

interaction with cellular adhesion molecules) and incorporation into the growing vessel or perivascular space (Urbich et al.; Shen et al., 2011).

The ability of MACs to home to and integrate at the site of angiogenic growth would also be an essential prerequisite for their participation in the process of B. henselae induced pathological angiogenesis in vivo. To investigate the ability of B. henselae infected MACs to associate with growing endothelium, MACs and HUVECs were stained red and green respectively and their interaction observed in a co-culture spheroid assay.

When MACs and HUVECs were combined at a ratio of 1:3, MACs incorporated successfully into the endothelial spheroid. Both infected and uninfected MACs were found to associate with growing vascular sprouts. MACs were found localized to both, tip and stalk areas of sprouts, indicating that B. henselae infection does not affect their ability to integrate into and interact with the growing endothelium.

Some indications exist that B. henselae infection may in fact increase the affinity of host-cells to the endothelium. In endothelial and epithelial cells, B. henselae infection has been shown to increase the expression of cellular adhesion molecules E-selectin, ICAM-1 and myeloid chemokine CCL2 (Fuhrmann et al., 2001; McCord et al., 2005). Furthermore, Mӓndle (Mändle, 2005) demonstrated that the infection of MACs with B. henselae also increases their chemoatracttant migration along an SDF-1 cytokine gradient.

The retention of infected MACs to sites of angiogenic growth and their intimate interaction with the active endothelium is also important in facilitating the transfer of pro-angiogenic effects to adjacent endothelial cells and the surrounding microenvironment.

Initial experiments indicated that infection of MACs with B. henselae resulted in activation of endogenous angiogenic response programs, increasing their angiogenic potential. To determine whether B. henselae infection also resulted in increased functional angiogenic effects, the rate of angiogenic sprouting was compared between co-culture spheroids composed of HUVECs mixed with infected or uninfected MACs. Indeed, in spheroids seeded with B. henselae infected MACs, the rate of endothelial sprouting increased significantly. Infected MACs induced an average of up to 1.61 fold more angiogenic sprouting in endothelial spheroids than uninfected cells indicating that the infection of MACs with B. henselae increases their pro-angiogenic activity and has significant functional effects on angiogenic growth of adjacent endothelium (Fig. 4.5A).

In previous studies the infection of THP-1 macrophages, HeLas and EA hy. 926 cells with B. henselae was found to induce the secretion of vasculoproliferative cytokines such as VEGF and CXCL8 and

conditioned medium from B. henselae infected THP-1 macrophages increased endothelial cell proliferation in vitro (Kempf et al., 2001; Resto-Ruiz et al., 2002; McCord et al., 2006). This has lead to the hypothesis that the infection of accessory cells such as myeloid and epithelial cells with B. henselae in vivo may contribute to pathological angiogenesis via the creation of a pro-angiogenic paracrine loop in which the release of pro-angiogenic cytokines stimulates proliferation of adjacent endothelium, attracts further myeloid cells and activates resident epithelial cells thus promoteing the progression of the B. henselae induced vascular tumor (Kempf et al., 2001; Kempf et al., 2002;

Resto-Ruiz et al., 2002).

A similar phenomenon is observed in malignant tumors where infiltrating myeloid cells (most prominently TAMs) are induced to secrete pro-angiogenic and immune regulatory cytokines, creating a pro-angiogenic tumor-microenvironment which stimulates the adjacent endothelium and contributes to tumor vascularization and invasion (Schmid and Varner, 2010).

To determine whether, the increased pro-angiogenic activity in B. henselae infected MACs was due to paracrine effects or physical interaction, further spheroid experiments were performed, placing infected MACs at varying degrees of separation from the endothelial spheroids. Spheroids were created purely from HUVECs and suspended in collagen gel. B. henselae infected MACs were either mixed with (Fig. 4.5B) or seeded onto the surface of collagen gel (Fig. 4.5C). In both conditions, the rate of sprouting angiogenesis increased dose-dependently when MACs were infected with B. henselae, indicating that no physical contact was necessary to transmit the pro-angiogenic effects of infected MACs.

Finally, conditioned medium from B. henselae infected MACs was used to culture HUVEC spheroids (Fig 4.5D). In this case, conditioned medium from B. henselae infected MACs showed a dose dependent effect and induced up to 6.71 fold more average sprouting than conditioned medium from uninfected MACs. These experiments indicate that paracrine effects are predominantly responsible for the pro-angiogenic activity of B. henselae infected MACs.

Overall, these results demonstrate that the infection of MACs with B. henselae not only results in the activation of angiogenic programs on a molecular level but increases their functional pro-angiogenic activity. Infection of cells did not inhibit their ability to home to and incorporate in growing vessels and had significant angiogenic effects on the surrounding endothelium.

In addition to demonstrating functional effects of B. henselae infection, results of the spheroid assay provides important clues about how B. henselae infection may affect the behavior of MACs in vivo.

The co-culture spheroid assay reproduces the interaction of MACs with a 3D sprouting endothelium

in vito and provides a realistic representation of the role of MACs as accessory cells in the process of angiogenic growth.

When exposed to angiogenic endothelium, B. henselae infected MACs incorporate into growing vascular sprouts and significantly increase angiogenic growth of the adjacent endothelium via predominantly paracrine factors. In the context of B. henselae associated vascular tumor formation, the interaction of infected MACs with the activated endothelium and parallel contribution to angiogenic growth through the release of paracrine factors would make a significant contribution to pathological angiogenesis and vascular tumor progression.

5.2.2 Matrigel capillary formation assay

To further elucidate the effects of B. henselae infection on MAC pro-angiogenic activity a second in vitro assay of angiogenic growth, the Matrigel capillary formation assay, was employed.

In the Matrigel assay, cells are seeded onto a 3D proteinacious matrix composed of mostly laminin and collagen IV. Mature endothelial cells such as HUVECs adhere to the matrix and over the course of 2-4h arrange into evenly spaced cell cluster connected by a web of “tubules” consisting of elongated endothelial cell branches 2-3 cells in length (Khoo et al., 2011).

In previous studies the infection of cultured endothelial cells with B. henselae has been shown to increases capillary structure formation in the Matrigel assay (McCord et al., 2006; Berrich et al., 2011).

In contrast to endothelial cells, MACs are not known to form spontaneous “capillary networks” in the Matrigel assay (Hur et al., 2004; Yoder, 2013a). Accordingly, when uninfected MACs were seeded in Matrigel culture they displayed no morphological differentiation, maintained spherical phenotypes, died and degraded over time (Fig. 4.6). B. henselae infected MACs, on the other hand, adhered strongly to the Matrigel matrix, developed long branching philopodia and gradually assembled into a complex network of chord structures that at times extend over the surface of the entire well plate (Fig. 4.6 and 4.7).

Although they display a similar basic composition and morphology to endothelial capillary networks, the chord networks developed by infected MACs in Matrigel culture differ from endothelial structures in two major ways.

In contrast to endothelial capillary networks, which consist of a dense web of thin chord-like connections 2-3 cells long, the chords networks formed by the B. henselae infected MACs were thicker, usually consisting of many directionally elongated and aligned cells. The intersecting chords

of heterogeneous length formed a sparser network and left larger interim areas mostly void of other cells.

In addition to differences in morphology, the capillary structures formed by B. henselae infected MACs differed from endothelial cells in the rate of structure assembly. While endothelial cell networks form in Matrigel cultures within 2-12h and degrade again shortly after, the capillary-like networks formed by the B. henselae infected MACs assembled gradually over the course of several weeks and were viable over long periods of time (41-77days). It is unclear why B. henselae infected MACs require such lengthy incubation times to fully develop the capillary-like network structures, however, it is likely that chord formation by B. henselae infected MACs is a more complex process that requires different cellular events. B. henselae infected MACs may first have to undergo some differentiation process, cellular activation or angiogenic re-programming before functional effects such as complex structure formation become apparent. The extended development time and long-term stability of the capillary structures formed by B. henselae infected MACs also highlights the anti-apoptotic effect of B. henselae infection. Uninfected MACs generally died out and degraded in culture after approximately 25 days (a typical lifespan of untreated primary human myeloid cells in culture) wheras B. henselae infected cells in the capillary-like structures were shown to be still viable at the end point of Matrigel cultures (d51) (Fig. 4.6 and Fig. 4.8).

Despite the difference in capillary-like structure formation between B. henselae infected MACs and endothelial cells, the ability of infected MACs to self-organize into such complex structures requires coordination of complex inter- and intracellular signaling networks, cellular migration, changes in morphology, and possibly remodeling of the extracellular matrix as well as the ability to resist apoptosis over long culture periods. This activity further underscores previous results indicating a significant angiogenic re-programming in B. henselae MACs leading to increased functional pro-angiogenic activity in infected cells.