**3. The application of MSCs and secretome in ischemic cardiac disease**

Cardiovascular diseases remain the leading cause of mortality and morbidity in worldwide. Various investigators have continued to assess a large number of cell types injected through several routes to promote cardiac repair in patients with cardiovascular diseases in both the preclinical and clinical stages. Clinical studies have largely been focused on the administration of MSCs [117, 118]. For instance, intracoronary injection of bone marrow MSCs led to an improved function of the left ventricle in subjects with acute myocardial infarction [119]. Mechanisms of action of MSCs administrated to the injured myocardium include accelerating angiogenesis process, diminished fibrosis, and regulation of immune response [102, 120]. Both in vitro and in vivo investigations have confirmed the trans-differentiating capacity of MSCs to effective cardiomyocytes in injured cardiac tissue [50]. In addition, documents revealed that MSCs from different sources release greater amounts of angiogenic factors (HGF, VEGF, and other growth factors), cell migration chemokine (SDF-1α), immune-signaling elements (IL-6, IL-8, and

MCP-1) TGF-β, neurotrophic factors (brain-derived neurotrophic factor (BDNF)), nitric oxide (NO), and improved cardiac restoration after injury [121].

Exosomes from MSCs exposed to hypoxia and FBS-free condition enhanced neovascularization in the injured heart [92, 122–124]. In a preclinical study, intramyocardial transplantation of exosome secreted from MSCs significantly improves blood flow rate and reduced infarct zone in the rat model [125]. Approximately, the entire small and large animal model of CVD preclinical investigations along with high-quality phase 0, I, II, and III clinical trials and meta-analysis studies vigorously confirmed that MSC therapy has the effective effects in developing angiogenic networks in ischemic regions [126, 127].

Ongoing researches on preconditioning and genetic manipulations of MSCs are needed to enhance angiocrine capacity governed by growth factors, microvesicles, microRNAs, long noncoding RNAs (lncRNAs), etc. [128, 129]. Finding the route of cell delivery, the optimum dose, the excellent cell source, and transplantation time are factors that still require to be addressed so as to achieve the aim of comprehensive cardiac regeneration.

## **4. Angiogenesis assays**

Both in vitro and in vivo angiogenesis assays are commonly used to investigate pro- and anti-angiogenic potential of stem cells and different cell types.

#### **4.1 In vitro analyses**

#### *4.1.1 Proliferation and survival assays*

Monitoring the proliferation of ECs is needed to develop microvascular units. Different survival and proliferation assays based on DNA synthesis or metabolic status are applicable. These assays could also predetermine the anti-angiogenic property of a specific compound in the context of tumor biology.

#### *4.1.2 Migration assays*

This method shows the migration in response to diverse factors, ability to digest basal membrane, and healing capacity of MSCs which is done by various assays as follows: Boyden chamber assay, Transwell® inserts, agarose assay, wound-healing assay, Teflon fence assay, phagokinetic track, etc. [130].

#### *4.1.3 Tube formation (tubulogenesis) assay*

This system is done in the 2D and 3D milieu and able to monitor alignment and juxtacrine connection of cells after plating on a specific substrate such as Matrigel, Fibrin, etc. Plated cells acquire phenotype to form capillary-like structures and lumen which are applicable to in vivo condition and evaluated in terms of tube area and number per microscopic field [130, 131].

#### *4.1.4 Aortic ring assay*

In this assay, the aorta from mouse or rats was removed and placed on collagen or fibrin matrix in serum-free condition. The angiogenic potential is determined by EC sprouting, polarized cells, and outgrowth appearance to the periphery [132].

**111**

*The Angiogenic Paracrine Potential of Mesenchymal Stem Cells*

the avascular area is monitored by the time [133].

*4.2.2 Chicken chorioallantoic membrane angiogenesis assay*

vessels from avian source to the implants were quantified [70].

The cornea is considered as avascular tissue with unique properties for monitoring the angiogenesis and done in the model of mouse, rat, and rabbit. In the procedure of corneal angiogenesis, the candidate biomaterials and polymer with putative pro- and anti-angiogenic factors were transplanted into the stromal pouch created by surgical approach. The penetration and ingrowth of nascent vessels into

This assay is performed on embryonated eggs by using polymer pellets and silastic rings containing target molecules on the surface of the chorioallantoic membrane. After the completion of distinct time, the number and dilation of blood

It is a choice of in vivo angiogenesis assay following administration of gelatinous protein mixture termed Matrigel into subcutaneous space. The target molecules could be administrated with Matrigel at the site of injection and systemically to the circulation system. To precisely elucidate the formation of de novo capillaries, fluorochrome agent could be administrated into the systemic circulation [130].

It is anticipated that MSC secretome and angiocrine could be used as an offthe-shelf alternative therapy to modulate angiogenesis/vascularization in distinct tissues. Considering both pro- and anti-angiogenic capacity, a big question remains to the identification of safety and efficacy of MSC secretome under specific conditions. Based on the data from different experiments, the angiogenic paracrine potential of MSCs is currently under investigation, and results of preclinical and translational studies, if confirmatory of previous basic experiments, could lead to human medicine for angiogenic modulation of tissues. The discovery of the signaling pathways that mastermind the paracrine pro- and anti-angiogenic potential of MSCs enables us to find appropriate policies for modulating angiogenic switch on/

All authors declared that there is no conflict of interest regarding the content of

*DOI: http://dx.doi.org/10.5772/intechopen.84433*

**4.2 In vivo analyses**

*4.2.3 Matrigel plug assay*

**5. Conclusion**

off in in vivo condition.

**Acknowledgements**

the current chapter.

*4.2.1 Corneal angiogenesis assay*
